Fermentation characteristics were measured and numbers and distribution by genera of ciliate protozoa were determined in ruminal fluid samples collected from 10 ruminally cannulated steers during the first 30 d of their being fed barley-based diets containing 62% (Medium Barley) or 95% (High Barley) barley grain (DM basis). Ruminal samples were collected at 5-d intervals over the 30-d periods beginning after adaptation (i.e., at the first full feeding of each diet). Ruminal pH and ammonia concentrations were lower (P < 0.001) with the High Barley than with the Medium Barley diet. Concentrations of total VFA and propionate and amylase activity of ruminal fluid were higher (P < 0.001) on High Barley than on Medium Barley. Total protozoal numbers in ruminal fluid were 42% lower (P < 0.05) on High Barley (470 × 103/mL) than on Medium Barley (804 × 103/mL). On Medium and High Barley diets, respectively, Entodinium spp. made up 89 and 91% of the ciliate protozoal populations. With the Medium Barley diet, relative proportions of Dasytricha, Ophryoscolex, Ostracodinium, Diplodinium, and Metadinium spp. in the total ciliate population were 4.5, 0.4, 0.5, 0.7, and 0.3%, respectively. When the High Barley diet was fed, these genera were not detected. In a subsequent survey, ruminal samples were collected from 200 finishing cattle at slaughter. Average protozoal population was 328 × 103/mL, and Entodinium spp. constituted 97% of the total. These data demonstrate that a large population of Entodinium spp. can persist in the rumen of cattle fed high barley grain-based finishing diets.