The effects of etomoxir, an inhibitor of mitochondrial long-chain fatty acid oxidation, on triacylglycerol metabolism in rat hepatocytes and adipocytes were investigated. Etomoxir inhibited the depletion of triacylglycerol stores in hepatocytes incubated without exogenous fatty acids and inhibited lipolysis in adipocytes. The effects on hepatocytes could be attributed to two mechanisms. At low concentrations (1–10 μM) R-etomoxir increased fatty acid esterification by inhibition of β-oxidation. This effect was specific for the R-enantiomer and was associated with increased triacylglycerol secretion. At higher concentrations (50–100 μM) RS-etomoxir inhibited lipolysis and triacylglycerol secretion, independently of inhibition of carnitine palmitoyltransferase I. These effects of RS-etomoxir on triacylglycerol metabolism and lipolysis may contribute to the chronic hypolipidaemic effects of etomoxir in vivo. © 1997 Federation of European Biochemical Societies.