Ketogenesis is the conversion of acetyl-CoA to the ketone bodies acetoacetate and beta-hydroxybutyrate (BHBA). In hepatic ketogenesis, which occurs during fasting in both nonruminant and ruminant animals, the source of acetyl-CoA is the mitochondrial oxidation of predominantly long-chain fatty acids. In the mature, fed ruminant animal, the ruminal epithelium is also capable of producing ketone bodies. In this case, the source of acetyl-CoA is the mitochondrial oxidation of butyrate produced by the microbial fermentation of feed. The purposes of this study were to determine ontogenic and dietary effects on ketogenic enzyme gene expression in developing lamb ruminal epithelium. Twenty-seven conventionally reared lambs and twenty-seven milk-fed lambs were slaughtered between 1 and 84 d of age. Six additional milk-fed lambs were weaned (the fed group) or maintained on milk replacer with a volatile fatty acid gavage (the VFA group) until 84 d of age. At slaughter, total RNA was extracted from samples of ruminal epithelium. The expression of the genes encoding acetoacetyl-CoA thiolase, the first enzyme in the ketogenic pathway, and 3-hydroxy-3-methylglutaryl-CoA synthase, the rate-limiting enzyme in the ketogenic pathway in nonruminant liver, were examined. Acetoacetyl-CoA thiolase and 3-hydroxy-3-methylglutaryl-CoA synthase mRNA concentrations increased with age independent of diet. 3-Hydroxy-3-methylglutaryl-CoA synthase mRNA levels in ruminal epithelium obtained from milk-fed lambs were low before 42 d of age, but a marked increase occurred by 42 d of age. At 84 d of age, there were no differences in acetoacetyl-CoA thiolase and 3-hydroxy-3-methylglutaryl-CoA synthase expression due to diet. The pattern of the expression of these genes, in particular, 3-hydroxy-3-methylglutaryl-CoA synthase, parallels the rate of production of BHBA by rumen epithelial cells isolated from the same lambs, which increased to conventionally reared adult levels at 42 d of age and did not differ with diet. In conclusion, development of the ketogenic capacity of the ruminal epithelium occurs as the animal ages, regardless of dietary treatment. Thus, the expression of the genes encoding the ketogenic enzymes are not affected by the presence of VFA in the ruminal lumen.