An epoxyalkane:coenzyme M (CoM) transferase (EaCoMT) enzyme was recently found to be active in the aerobic vinyl chloride (VC) and ethene assimilation pathways of Mycobacterium strain JS60. In the present study, EaCoMT activity and genes were investigated in 10 different mycobacteria isolated on VC or ethene from diverse environmental samples. In all cases, epoxyethane metabolism in cell extracts was dependent on CoM, with average specific activities of EaCoMT between 380 and 2,910 nmol/min/mg of protein. PCR with primers based on conserved regions of EaCoMT genes from Mycobacterium strain JS60 and the propene oxidizers Xanthobacter strain Py2 and Rhodococcus strain B-276 yielded fragments (834 bp) of EaCoMT genes from all of the VC- and ethene-assimilating isolates. The Mycobacterium EaCoMT genes form a distinct cluster and are more closely related to the EaCoMT of Rhodococcus strain B-276 than that of Xanthobacter strain Py2. The incongruence of the EaCoMT and 16S rRNA gene trees and the fact that isolates from geographically distant locations possessed almost identical EaCoMT genes suggest that lateral transfer of EaCoMT among the Mycobacterium strains has occurred. Pulsed-field gel electrophoresis revealed large linear plasmids (110 to 330 kb) in all of the VC-degrading strains. In Southern blotting experiments, the strain JS60 EaCoMT gene hybridized to many of the plasmids. The CoM-mediated pathway of epoxide metabolism appears to be universal in alkene-assimilating mycobacteria, possibly because of plasmid-mediated lateral gene transfer.