Methane (CH4) production by rumen methanogens lowers hydrogen (H2) pressure and, in theory, prevents inhibition of fermentation processes by H2 accumulation. The present study aimed at examining effects of short-term H2 infusion on CH4 production and the volatile fatty acid (VFA) profile. Four lactating Holstein dairy cows fitted with rumen cannula were each infused once with pure H2 into the rumen at a rate of 48.0 L/h during 5.75 h in between the morning and afternoon feeding. Gas exchange and feed intake were measured continuously by open-circuit respiration chambers during 5 days. Rumen liquid was sampled twice a day in connection with milking and feeding (0630 hours and 1700 hours) and analysed for VFA. Gas exchange and dry matter intake (DMI) were analysed for 5-h steady-state H2 concentrations (TI5) measured in respiration chambers and for 24-h time intervals (TI24) on the day before, during and after infusion. Hydrogen infusion did not affect the total VFA concentration and VFA molar proportions for either time interval. Methane production was higher for TI5 during infusion (130 L/5 h) than it was the day before infusion (120 L/5 h), but not the day after infusion (122 L/5 h). Methane production for TI24 and DMI for TI5 and TI24 were unaffected. Oxygen consumption and CH4 : CO2 were highest during infusion for TI5, but not for TI24. After correcting for H2 naturally produced, on average, 46.7 L H2/h was measured during TI5, indicating that 2.7% of the infused H2 was retained in the rumen. In conclusion, H2 infusion did not affect the VFA profile, but slightly increased CH4 production and CH4 : CO2.