The assay conditions for palmitoyl-CoA synthetase (P-CoA S) and carnitine palmitoyltransferase (CPT) in homogenates of human blood platelets have been studied. The assay based on trapping of palmitoyl-CoA by carnitine in the presence of exogenous CPT gave higher activity of P-CoA S than the assay based on direct isolation of the palmitoyl-CoA formed. The activity of CPT was higher on exogenous palmitoyl-CoA than on endogenous palmitoyl-CoA formed from palmitic acid and CoA in the presence of endogenous P-CoA S. The activity of CPT was strongly dependent on the incubation time and the amount of platelets used. The initial activity of this enzyme in human blood platelets was higher than previously assumed.