AIADMK coordinator O Panneerselvam and co-coordinator K Palaniswami announced "removing" 44 functionaries from the party's primary membership.
Those who faced the action included R Samy, a former MLA from Melur in Madurai and a staunch supporter of Dhinakaran.
In a joint statement, Panneerselvam and Palaniswami named the functionaries, holding various posts including membership to the powerful decision making General Council and covering the AIADMK units in Madurai, Villupuram, Dharmapuri, Tiruchirappalli and Perambalur among others.
"Party workers are requested not to have any truck with them," Palaniswami, also the Chief Minister, and his deputy Panneerselvam, said in the statement.
Further, Panneerselvam and Palaniswami "relieved" two others from party posts in Puthukottai and Vellore districts.
Stung by the R K Nagar bypoll defeat, the ruling AIADMK on December 25 cracked the whip against nine party functionaries.
The two leaders had then sacked four of Dhinakaran's aides as district secretaries, while expelling five others.
The bypoll, necessitated due to the death of late Chief Minister J Jayalalithaa who was the sitting MLA, was seen as a litmus test for the 20-month-old AIADMK government.
Dhinakaran, sidelined by the ruling camp a few months ago, had won as an independent candidate.
He had earlier lost his battle for the party symbol 'Two Leaves' to the Palaniswami-led ruling camp and contested the election on Pressure Cooker symbol.
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