Aims: To investigate the relationship between the protonation of medium-chain fatty acids (MCFA) and their inhibitory effect on a ruminal methanogen species. Methods and Results: Cell suspensions of Methanobrevibacter ruminantium M1 in 1 mg dry matter (DM) ml(-1) were supplemented with lauric acid (C-12) and myristic acid (C-14) at a concentration of 8 mu g ml(-1) with different pH levels of the potassium-free buffer, where the calculated degrees of protonation of C-12 and C-14 varied from 0.3 to 50% and from 1 to 76% respectively. Methane formation, ATP efflux, potassium leakage and cell viability were monitored 15, 30 and 45 min after the reaction started. Declining methane formation rate, increasing ATP efflux and potassium leakage, and decreasing survival of M. ruminantium were observed with increasing degrees of protonation, i.e. with decreasing pH. Conclusions: The inhibition of methanogenesis by C-12 and C-14 is more efficient at a pH of 5-6 as compared to pH 7. Significance and Impact of the Study: Methane mitigation strategies in ruminants which use supplementation of feed with MCFA such as C-12 and C-14 may be more effective in a low rumen pH environment. This finding is helpful in designing diets to effectively decrease methane emissions by ruminants.