The influence of fasting/refeeding and insulin treatment on ketogenesis in 12-day-old suckling rats was studied in intestine and liver by determining mRNA levels and enzyme activity of the two genes responsible for regulation of ketogenesis: carnitine palmitoyl transferase I (CPT I) and mitochondrial HMG-CoA synthase. Fasting produced hardly any change in mRNA or activity of CPT I in intestine, but led to a decrease in mitochondrial (mit.) HMG-CoA synthase. In liver, while mRNA levels and activity for CPT I increased, neither parameter was changed in HMG-CoA synthase. The comparison of these values with the ketogenic rate of both tissues under the fasting/refeeding treatment shows that HMG-CoA synthase could be the main gene responsible for regulation of ketogenesis in suckling rats. The small changes produced in serum ketone bodies in fasting/refeeding, with a profile similar to the ketogenic rate of the liver, indicate that liver contributes most to ketone body synthesis in suckling rats under these experimental conditions. Short-term insulin treatment produced increases in mRNA levels and activity in CPT I in intestine, but it also decreased both parameters in mit. HMG-CoA synthase. In liver, graphs of mRNA and activity were nearly identical in both genes. There was a marked decrease in mRNA levels and activity, resembling those values observed in adult rats. As in fasting/refeeding, the ketogenic rate correlated better to mit. HMG-CoA synthase than CPT I, and liver was the main organ regulating ketogenesis after insulin treatment. Serum ketone body concentrations were decreased by insulin but recovered after the second hour. Long-term insulin treatment had little effect on the mRNA levels for CPT I or mit. HMG-CoA synthase, but both the expressed and total activities of mit. HMG-CoA synthase were reduced by half in both intestine and liver. The ketogenic rate of both organs was decreased to 40% by long-term insulin treatment. The different effects of refeeding and insulin treatment on the expression of both genes, on the ketogenic rate, and on ketone body concentrations are discussed.