The effect of 7 individual AA (Leu, Met, Phe, Trp, Val, His, and Lys) on ruminal gas production and fermentation was investigated in a batch culture in vitro experiment. All AA were examined at 2 levels: 20 and 60 mg/L incubation medium. Two separate incubations were conducted for 6 (group 1) or 12 h (group 2). Rumen inoculum to buffer ratio was 1: 2. Gas pressure was recorded and a gas sample analyzed for concentration every 3 h for both groups. Compared with blank (no AA), gas production was lower (P ≤ 0.03) for Phe, Trp, Val, His and Lys (ranging from 48.2 to 57.6 mL vs. 60.7 mL). Gas production was not affected by treatment in group 2 (P ≥ 0.30). Compared with blank (6.38%), all 7 AA had lower methane concentration in the gas phase (ranging from 4.61 to 5.66%, P ≤ 0.02) in group 1. Methane concentration was also lower (ranging from 4.36 to 4.66% vs. 4.97% for the blank, P ≤ 0.03) in group 2, excepted for His (4.76%) and Met (5.12%). Total VFA concentration did not differ between treatments (P = 0.45). In group 1, isobutyrate concentration was increased by Phe (0.32 µmol/mL, P ≤ 0.001) compared with blank (0.23 µmol/mL), and in group 2, isobutyrate (0.25 µmol/mL, P = 0.04) and isovalerate (0.47 µmol/mL, P = 0.05) concentrations were decreased by His compared with blank (0.34 and 0.51 µmol/mL). Other individual VFA were not affected by treatment. Compared with blank (1.96 mmol/L), ammonia concentration was greater (average 2.31 mmol/L, P ≤ 0.002) for the AA treatments in group 1. In group 2, AA decreased ammonia concentration (average 1.42 mmol/L), compared with blank (2.20 mmol/L), except for His and Leu, 1.71 and 1.77 mmol/L, respectively. Protozoal counts were lower for AA treatments, except for His, (ranging from 1.87 to 2.77 × 10⁴/mL, P < 0.001) compared with the blank (4.10 and 2.90 × 10⁴/mL for group 1 and 2, respectively). These results indicate that some AA may have an inhibitory effect on total gas, methane production and some individual VFA in an in vitro batch culture system, but do not affect total VFA production and have a variable effect on ammonia concentration, depending on dose and incubation time.