A fluorometric assay for the determination of hepatic carnitine palmitoyltransferase (CPT) activity was slightly modified for use with cattle samples. With this assay, the K-m value was 0.56 +/- 0.10 mM with respect to L-carnitine (mean +/- SD, n=4) and was 9.6 +/- 2.2 mu M (n=3) with respect to palmitoyl-CoA. The average hepatic CPT activity was 33.6 +/- 2.0 mu mol CoASH/min/g protein in 38 healthy cattle and was similar in both sexes and among breeds. Hepatic CPT activity showed no correlation with serum phospholipid, free fatty acid, triglyceride or total cholesterol concentrations.