After incubating isolated rat hepatocytes with [1-14 C]palmitic acid, CoA and ATP (+MgCl2), a significant amount of [1-14 C]palmitoyl-CoA was found in the incubation medium. There was no correlation between its rate of synthesis and the degree of intactness of the cells. The results indicate that there is a long-chain fatty acyl-CoA synthetase active on the external surface of the hepatocyte plasma membrane. The activity of this enzyme was negligible in primary cultures of rat hepatocytes, suggesting that the exofacial long-chain acyl-CoA synthetase is an artifact of the collagenase perfusion technique used to prepare the hepatocytes.