The effects of yeast Saccharomyces cerevisiae, either inactivated (by osmotic pressure, designated IY) or provided as a culture containing live yeast cells (YC), on ruminal fermentation of two different diets were investigated in vitro. Total mixed rations (TMR) having forage:concentrate ratios of 0·6:0·4 (medium–high forage diet) and 0·2:0·8 (low-forage diet) were incubated in batch cultures of mixed ruminal micro-organisms to which either IY (to reach concentrations of 500 and 250 mg product/l incubation medium) or YC (at a concentration of 150 mg product/l) were added directly as powder. To evaluate the effects of the additive on ruminal microbial population, sheep used as donors of rumen fluid were allocated to three experimental groups: Control (no additive), IY and YC, that received a diet with the corresponding additive for 10 days. With both diets, YC decreased ruminal pH compared to control, whereas IY had no effect. Adding yeast products to the high-fibre diet affected total volatile fatty acid (VFA) production and VFA composition, in general with a slight increase in IY and a significantly greater increase in response to the addition of YC. Ammonia nitrogen (P = 0·006), total gas production (P < 0·001) and in vitro dry matter disappearance (IVD) (P < 0·001) showed the highest values with YC. Methane production was higher than the control when the IY inoculum was used, and increased even more with the YC inoculum (P < 0·001). With the high-concentrate TMR, no effects on total VFA concentration were observed when yeast additives were used. Similar trends were shown for lactate and methane production and total gas production, where values tended to be higher when using the YC inoculum (P values of 0·055, <0·001, 0·006 and <0·001, respectively). After 144 h of incubation, differences were observed only with the high-fibre diet in the cumulative gas production at 24 h of incubation and in the average fermentation rate, which was greater with YC, although the asymptotic gas production was not affected. These results indicate that live yeasts affect ruminal fermentation slightly more than inactivated yeasts, although both products require a regular administration and some adaptation of the ruminal microbial population for the stimulatory effects to become apparent. The effects of yeasts on ruminal fermentation are diet-dependent, being more noticeable with a high-fibre substrate, and subtle with a high-concentrate diet.