Clostridium thermocellum cell extracts exhibit specific endonuclease activity with very little non-specific exonuclease activity at 55 degrees C, The Dam methylation system of Escherichia coli offers complete protection from digestion by C. thermocellum ATCC 27405 cell extracts for all DNA tested(totaling > 100 kb, insuring that most potential restriction sequences have been exposed), Based on both the Dam recognition sequence and the similarity of cell extract and MboI DNA digests, the C. thermocellum restriction enzyme recognition sequence appears to be 5’ GATC 3’. Cell extracts made from a second thermophile, C. thermosaccharolyticum ATCC 31960 do not exhibit specific endonuclease activity under the conditions tested. Genomic DNA from C. thermocellum exhibits a Dam(+) phenotype while genomic DNA from C. thermosaccharolyticum exhibits a Dam(-) phenotype.