Starved male weanling rats supplemented with 20 mmol/L pivalate in their drinking water exhibit significantly depressed concentrations of carnitine in tissues and plasma. In addition, pivalate supplementation has been linked with increased renal and hepatic trimethyllysine hydroxylase (TMLH) activity, whereas carnitine supplementation has been associated with significantly decreased hepatic gamma-butyrobetaine hydroxylase (BBH) activity. The purpose of this study was to determine whether pivalate or carnitine supplementation affects the activity and genetic expression of 2 enzymes of carnitine (Cn) biosynthesis, TMLH and BBH, expressed as mRNA abundance, relative to the abundance of beta-actin mRNA. Male weanling rats were administered the control treatment (C; n = 6), the pivalate treatment (P; n = 7), or the pivalate treatment plus supplemental dietary carnitine (P+Cn; n = 7). Rats in group P had elevated renal TMLH activity, relative to the other groups (P < 0.05). The groups did not differ in the abundance of renal or hepatic TMLH or BBH mRNA. A previously unreported finding was the quantifiable level of renal BBH mRNA, which was verified by direct sequencing of the BBH cDNA product amplified from kidney RNA. The groups did not differ in renal BBH mRNA abundance and renal BBH enzyme activity was not detected. Thus, the alterations in enzyme activities in the pivalate-treated rats are not regulated at the transcriptional level, and are apparently related to post-transcriptional effects on the enzymes themselves.