Effects of Propionibacterium acidipropionici P169 on gas, methane (CH 4 ) production and volatile fatty acids in in vitro rumen cultures were studied. Ground barley (Experiment 1), mixed forage (Experiment 2) or mixed concentrate-forage (Experiment 3) served as substrates. Three doses of P169 were used in Experiments 1 and 2 [1.2 Â 10 4 ; 1.2 Â 10 6 ; 1.2 Â 10 8 colony forming unit (CFU)/mL], while two doses (2.4 Â 10 6 ; 2.4 Â 10 8 CFU/mL) were used in Experiment 3. In Experiment 1, P169 increased (P < 0.05) gas production at 6-12 h and CH 4 (mL/g diet dry matter) at all times. In Experiment 2, CH 4 was increased at 6 h (P < 0.01). At 12 h, propionate declined (P < 0.01) in Experiment 1 and increased at 48 h in Experiment 3. The inability of P169 to increase propionate and its lack of persistence in mixed ruminal cultures may account for its inability to alter CH 4 production.