The objective of Exp. 1 was to compare the effects of virginiamycin (VM; 0, 175, or 250 mg x animal(-1) x d(-1)) and monensin/tylosin (MT; 250/ 90 mg x animal(-1) x d(-1)) on ruminal fermentation products and microbial populations in cattle during adaptation to an all-concentrate diet. Four ruminally cannulated, Holstein steers were used in a 4x4 Williams square design with 21-d periods. Steers were stepped up to an all-concentrate diet fed at 2.5% of BW once daily. Ruminal pH, protozoal counts, and NH3-N and VFA concentrations generally were unaffected by VM or MT. Mean counts of Lactobacillus and Streptococcus bovis were lower (P<.05) for VM-treated compared with control or MT-treated steers. Both VM and MT prevented the increase in Fusobacterium necrophorum counts associated with increasing intake of the high-concentrate diet observed in the control. The objective of Exp. 2 was to compare the effects of VM and MT on ruminal pH, L(+) lactate and VFA concentrations, and F. necrophorum numbers during carbohydrate overload. Six ruminally cannulated Holstein steers were assigned randomly to either the control, VM (175 mg/d), or MT (250 + 90 mg/d) treatments. Acidosis was induced with intraruminal administration of a slurry of ground corn and corn starch. The VM and MT premixes were added directly to the slurry before administration. Carbohydrate challenge induced acute ruminal acidosis (pH was 4.36 and L (+) lactate was 19.4 mM) in controls by 36 h. Compared with the controls, steers receiving VM or MT had higher (P<.05) ruminal pH, and the VM group had a lower (P<.05) L (+) lactate concentration. Fusobacterium necrophorum numbers initially increased in VM- and MT-administered steers. In the control steers, F. necrophorum was undetectable by 36 h. Virginiamycin seemed to control the growth of ruminal lactic acid-producing bacteria and, therefore, has the potential to moderate ruminal fermentation in situations that could lead to rapid production of lactic acid.