Twelve ruminally and duodenally cannulated beef steers (initial BW 368 +/- 25.3 kg) and four ruminally cannulated beef heifers (initial BW 559 +/- 79.5 kg) were used to evaluate the effects of yeast culture (YC) and advancing season on ruminal fermentation, microbial protein synthesis, ruminal fluid kinetics, and site of digestion. Treatments were control and YC supplementation (28.4 g.steer-1.d-1 dosed ruminally). Steers grazed from late June to early November 1991 on mixed-grass prairie. Ruminal pH decreased (P < .10) from late July to early October. Fluid dilution rate decreased (P < .10) as the grazing season advanced, whereas ruminal fluid volume and flow rate increased (P < .10) from late July to early October. Ruminal ammonia concentration (milligrams/deciliter) was lower (P < .10) during late July and late August than during late June and early October at 4, 8, 12, and 16 h after sunrise in YC-supplemented steers. Molar proportions of propionate and butyrate were greater (P < .10) in control than in YC-supplemented steers at 0, 4, 12, and 24 h and 0, 4, and 12 h after sunrise, respectively, during late July. Acetate (mol/100 mol) was greater (P < .10) during late July and late August than in late June and early October for YC-supplemented steers at 0, 4, and 8 h after sunrise. True ruminal OM digestion was greatest (P < .10) in late June, intermediate in late July and late August, and least in early October. Supplementation with YC increased (P < .04) true ruminal OM digestion in late June and late July. Steers receiving YC had greater (P < .07) duodenal bacterial N flow in late July. These data indicate that yeast culture supplementation can increase true OM digestibility early in the grazing season. Advancing season seems to result in increased ruminal fluid volume, lower true ruminal OM digestion, and greater microbial efficiency.