Technical Abstract: When mixed ruminal bacteria (approximately 400 mg protein ml-1) from a cow fed timothy hay were incubated in vitro with carbon dioxide and hydrogen (0.5 atm) for less than 8 h, the first order rate of methane production was 17 µmol ml-1. Semi-purified bacteriocin from Streptococcus bovis HC5 (bovicin HC5) inhibited methane production, by as much as 50%, and even low concentration of bovicin HC5 (128 activity units ml-1) caused a significant decrease. Mixed ruminal bacteria that were transferred successively retained their ability to produce methane from carbon dioxide and hydrogen, and the first order rate of methane production did not decrease. Cultures that were treated with bovicin HC5 (128 activity units ml-1) gradually lost their ability to produce methane, and methane was not detected after 4 transfers. These latter results indicated that ruminal methanogens could not adapt and become resistant to bovicin HC5. When the chromosomal DNA that was amplified with 16S rDNA primers specific to archaea, digested with restriction enzymes (Hha I and Hae III) and separated on agarose gels, approximately 12 bands were observed. DNA from control and treated cultures (3rd transfer) had the same banding pattern, and this result indicated bovicin HC5 was not selective. Given the perception that the routine use of antibiotics in animal feeds should be avoided, bacteriocins.