Four rumen-fistulated steers, randomly assigned to two groups (control and salinomycin fed) were used to monitor the changes in rumen microbial populations and volatile fatty acids (VFA) concentrations associated with feeding salinomycin (0.22 mg X kg-1 X day-1). Steers were adapted to an alfalfa hay and grain (80:20) diet before supplementing the diet with salinomycin, and then the diet was changed to 50:50 and 20:80 ratios of alfalfa hay to grain at 2-week intervals. Rumen samples for total and selective enumeration of anaerobic bacteria. VFA analysis, and enumeration of protozoa were collected during the 80:20 alfalfa hay-to-grain diet before salinomycin feeding, and during the 80:20, 50:50, and 20:80 hay-to-grain diets with salinomycin. At each sampling period, rumen samples were collected at 3 h after feeding on three consecutive days. Salinomycin feeding had no effect on rumen pH and total VFA concentration. The acetate-to-propionate ratio was significantly lower in salinomycin-fed steers than in the control. The molar proportion of butyrate increased in both control and salinomycin-fed steers. Total anaerobic bacterial counts were lower in salinomycin-fed steers than in the control steers after 8 weeks of salinomycin feeding. Salinomycin-resistant bacteria increased from 7.6 to 15.6% in salinomycin-fed steers but remained unchanged in control steers. Salinomycin had no effect on cellulolytic and lactate-utilizing bacteria, but the proportion of amylolytic bacteria was higher in salinomycin-fed steers than in control steers. The total number of protozoa decreased initially in salinomycin-fed steers. The initial reduction was due to reduced numbers of Entodinium species. Holotrichs were unaffected by salinomycin feeding.