The 92-year-old wheelchair-bound DMK leader appeared before the court.
Principal Sessions Judge N Audhinathan adjourned the hearing in the case till March 10.
Karunanidhi had recently said that this was "one among the many cases filed against me" by the Jayalalithaa government and he would face them legally.
A large number of DMK cadres thronged the court premises with police making tight security arrangements to prevent any untoward incidents. DMK party lawyers were also present.
Karunanidhi, who enjoys 'Z' category security status, was escorted by a posse of security men.
Top DMK leaders including his son and party Treasurer, M K Stalin, daughter and Rajya Sabha MP, Kanimozhi, Arcot Veerasami, grand nephew Dayanidhi Maran and T R Baalu were present in court.
Principal Sessions Judge N Audhinathan had last month directed Karunanidhi to appear before the court on January 18 in connection with an alleged defamatory article published in the party mouthpiece Murasoli in November about the All India Anna Dravida Munnetra Kazhagam government's four-year rule.
The court had also summoned Murasoli's Editor, Printer and Publisher S Selvam to appear on the same day in this regard.
The issue pertains to Karunanidhi quoting from an article in Tamil bi-weekly Ananda Vikatan's November 25, 2015 issue on the matter.
Writing in party mouthpiece Murasoli, the DMK chief had cited the article which had made a critical assessment of the four year-old AIADMK government's performance.
The party's legal wing secretary, R Girirajan, had asked his party lawyers to be present in the principal sessions court in good numbers, in an apparent show of strength.
Meanwhile, replying to a question after coming out of the court, Karunanidhi told reporters, "I will not compel other political parties to form an alliance for the coming assembly polls. The decision to form any alliance should be their own."
The state assembly polls are scheduled in May.
The rare court appearance by Karunanidhi, just months ahead of assembly elections in Tamil Nadu, is being seen as a shot in the arm for the party by its cadres.
Some party leaders said it had "infused new blood" in the party which is looking to stage a comeback after back-to-back poor showing in the last assembly and Lok Sabha elections.
"He (Karunanidhi) has appeared in many cases. Today he appeared before (the Principal Sessions court) to tell the people what is going on," DMK Organising Secretary R S Bharathy said.
However, the party chose not to link it with the coming polls and said their 'Thalaivar' (leader) was only trying to tell the people what was happening in the AIADMK rule.
Bharathy was referring to a number of defamation cases filed against DMK and other political parties by the Jayalalithaa government.
Asked if Monday's move was aimed at preparing the party workers for the polls, Bharathy replied in the negative and said that defamation cases had been filed by the government even against certain media houses.
Another leader, T R B Raja, the party's MLA from Mannargudi, also insisted that Karunanidhi's court appearance had nothing to do with elections, but however maintained it had "infused new blood", especially among the younger cadre.
He said that it if a 92-year-old leader can do something like appearing before a court and sending a message to the party, then the many number of young workers can do anything constructively.
"This has got nothing to do with the polls. It proves that he can face anything, politically and legally. However, this has infused a new blood, especially to the many young party workers," he said.
Some other party workers also expressed enthusiasm following Karunanidhi's decision not to seek exemption and to appear in the court.
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