Many authors agree that alteration of the energy substrate from glucose to free fatty acid occurs during the early stage after partial hepatectomy. An accelerative effect of carnitine on the early phase of liver regeneration was suggested in several reports, but much controversy prevails. Using male Wistar rats weighing about 200 g as subjects, we undertook partial hepatectomy with resection of the median and left lateral lobes (67%). Another group of rats undergoing a sham operation was compared. Rats were killed at 6, 24, 48, or 72 hr after the operation. The rate of synthesis of DNA and content of DNA in remnant liver were chosen as regenerative indicators. Serum carnitine, free fatty acid and its metabolites, remnant liver carnitine palmitoyltransferase I (CPT-I) activity, high-energy phosphate (HEP), including adenosine triphosphate (ATP), and creatine phosphate (CP) were measured. The results showed a marked decrease of HEP, ATP, and CP with suddenly increased free fatty acid and total ketone body in serum that occurred during the early regenerating phase after partial hepatectomy. Serum L-carnitine also increased markedly in this early stage. The mitochondrial CPT-I activity in the remnant liver decreased significantly 24 hr after partial hepatectomy. Our data show that regenerating liver utilizes free fatty acids as an immediate main substrate. Mitochondrial respiration with a CPT-I effect could be an important reaction in this utilization.