Proglycosyn (LY177507) belongs to a series of powerful agents that stabilize liver glycogen stores by promoting glycogen synthesis from different precursors and inhibiting glycogenolysis and glycolysis. In the present study we have examined the effects of proglycosyn on fatty acid metabolism in isolated hepatocytes. Preincubation of hepatocytes with medium containing proglycosyn led to a marked inhibition of fatty acid synthesis de novo and acetyl-CoA carboxylase activity without affecting fatty acid synthase. Likewise, proglycosyn depressed the synthesis of triacylglycerols and phospholipids from labeled palmitate. Although octanoate oxidation was unaffected by proglycosyn, mitochondrial palmitate oxidation was notably stimulated. This effect may be attributed to the proglycosyn-induced decrease of intracellular malonyl-CoA levels relative to control incubations and the concomitant relieve of the inhibition of the mitochondrial-outer-membrane carnitine palmitoyl-transferase by malonyl-CoA. By contrast, neither peroxisomal palmitate oxidation nor peroxisomal carnitine palmitoyltransferase activity was changed upon hepatocyte incubation with proglycosyn. Results thus indicate that proglycosyn increases the fatty-acid-oxidation efficiency of the liver at the expense of lipogenesis, and this may contribute to the proglycosyn-induced sparing of liver glycogen stores.