In total, 73 strains of methanogen isolates from our laboratory and 6 from culture collections were examined for the presence of plasmid DNA. Five strains were found to contain detectable plasmids. Multiple plasmids were found in two isolates, while three strains contained only one plasmid each. A physical map of the plasmid pT3 was constructed by use of six different restriction endonucleases. All sites were aligned with a single BglI site, and the position of the restriction sites was determined by double or sequential digestion of the plasmid DNA.