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To investigate the presence of GATC-specific modification and restriction activities in rumen isolates of Treponema sp.The presence of N6-methyladenine within GATC (Dam) sequences was analysed using isoschizomeric restriction endonucleases having different sensitivities to the methylation of the target sequence. A fast screening method was used for testing of site-specific endonuclease activities directly in crude cell extracts. Three out of six rumen isolates of Treponema sp. showed restriction activities. Restriction endonucleases were further purified by Heparin-Sepharose chromatography. Using PCR and specific primers, no sequence homologous to the T. pallidum dam gene was found.Three rumen treponemal strains were documented to possess MboI isoschizomeric restriction– modification systems.This is the first report on restriction activity in rumen treponemes.