Experiments were conducted to investigate the role of the protein matrix in the digestion of barley and corn by ruminal microorganisms. Grains, ground and collected on sieves as two particle fractions .25 to .89 mm (small particles) and 2.00 to 3.00 mm (large particles), as well as isolated barley and com starch granules, were incubated with ruminal inocula. For both grains, digestion of starch in small particles was greater (P < .001) than that of large particles. At 16 and 24 h starch digestion was greater (P < .01) in barley than in com, for small and large particles. Digestion of barley starch granules did not differ (P > .05) from that of corn starch granules at any incubation time. A 4-h preincubation of small-particle corn and barley with protease increased (P < .001) microbial digestion of starch in corn at 16 h but did not affect digestion of barley. When four ruminally cannulated steers were fed diets containing 80% barley, corn, or wheat or 100% alfalfa hay in a 4 x 4 Latin square experiment, amylolytic activity of ruminal inoculum was higher for steers fed grains than for those fed alfalfa. However, when standardized for total viable counts of bacteria, ruminal amylolytic activity did not differ (P > .05) among diet treatments, but proteolytic activity of ruminal inoculum for steers fed alfalfa was higher (P < .01) than that for steers fed cereal grains. Activity of serine proteases in ruminal inoculum was higher (P < .05) for steers fed alfalfa than for steers fed grains. The protein matrix seemed to be the major factor responsible for differences in ruminal digestion of ground corn and barley. Differences in the properties of the protein matrix among cereal grains did not alter ruminal amylolytic or proteolytic activity.