Twenty calves (7 mo old) were blocked by breed, sex, and weight into five groups of four calves and randomly assigned to either a 90% forage (alfalfa) or a 90% grain (50% sorghum and 50% wheat) diet fed at one (1M) or two (2M) times NEm for 140 d. Samples of ruminal epithelial tissue were collected from the anterior ventral sac, and papillae were cut free by hand and used for in vitro incubations and acyl-CoA synthetase assays. Substrates were acetate (90 mM), propionate (60 mM), butyrate (30 mM), and glucose (20 mM). Net productions of beta-hydroxybutyrate and acetoacetate from acetate were greater (P less than .01) with the 2M feeding; however, 14CO2 production from acetate was greater (P less than .05) with the grain diet. Net production of lactate (P = .09) and pyruvate (P less than .01) from propionate increased with the 2M feeding, whereas net lactate production from glucose decreased (P less than .01). Uptakes of VFA were similar with 1M and 2M feeding and were about 10-fold greater than uptakes of glucose. Production of 14CO2 from propionate was two- to fivefold greater than from acetate, butyrate, or glucose. Oxygen consumption was greater (P less than .01) with 2M feeding and unaffected by substrate. Activities of butyryl-CoA synthetase (nmol.mg of tissue-1.h-1) were greater (P less than .05) for animals consuming the forage diets. Addition of butyrate inhibited acetyl- and propionyl-CoA synthetase activity by 63 and 92%, respectively for all dietary treatments. Overall, metabolism of ruminal epithelial tissue tended to increase with the 2M feeding. Influence of dietary forage content on metabolism and activity of acyl-CoA synthetases was minimal, but the high-forage diet caused slight increases.