Riboflavin deficiency leads to depressed mitochrondrial fatty acid oxidation rates but increased activity of carnitine palmitoyltransferase (CPT). Starvation leads to increased CPT activity in ad libitum-fed, riboflavin-supplemented rats. The present studies examined the mechanism of the increase in CPT activity in riboflavin deficiency and whether it was additive to that seen in starvation. Rats were divided into three groups initially: riboflavin-sufficient, ad libitum-fed; riboflavin-deficient, ad libitum-fed; and pair-fed. These groups were subdivided after 5 wk into fed and 24- and 48-h starved groups. When riboflavin-deficient rats were starved for 24 or 48 h, there was only 30-40% increase in hepatic CPT activity, in contrast to the ad libitum-fed, riboflavin-supplemented rats, in which activity increased twofold. CPT activity of pair-fed rats was similar to that of controls in fed state and did not increase significantly with starvation. CPT translation, mRNA levels and transcription rates correlated with CPT activity, as did immunoreactive CPT. Concurrently, hepatic ketone production and plasma β-hydroxybutyrate concentration increased during starvation in the control and pair-fed but not in the riboflavin-deficient rats. The results indicate that increased CPT activity in riboflavin deficiency and starvation results at least in part from increased synthesis. Furthermore, the data support previous work suggesting that the block in fatty acid oxidation occurs in the β-oxidation pathway at the level of acyl-CoA dehydrogenases.