The effects of vasoactive intestinal peptide (VIP) on fatty acid oxidation in isolated rat enterocytes were investigated. VIP (10(-7) M) increased more than 2-fold the production of 14CO2 from [U-14C]palmitate. This effect was dose-dependent (K0.5 = 5.10(-11) M) and appeared to be related to the stimulation of cAMP production since it was mimicked by forskolin (10(-4) M). VIP also stimulated oxygen consumption of the cells, an effect accounted for by the stimulation of the oxidation of both exogenous added palmitate (0.12 mM) and endogenous fatty acids produced by lipolysis. VIP appeared to specifically enhance the oxidation of long-chain fatty acids since its effects were counteracted by 5.10(-5) M sodium 2-[6-(chlorophenoxy)hexyl]oxirane-2-carboxylate, a potent inhibitor of carnitine palmitoyltransferase 1, and since VIP did not affect cell respiration in the presence of octanoate. These results suggested that VIP stimulated long-chain fatty acid oxidation by increasing their translocation into the mitochondria. Therefore, we examined the effect of VIP on the activity of acetyl-coenzyme A carboxylase, the enzyme responsible for the biosynthesis of malonyl-CoA, a physiological inhibitor of carnitine acyltransferase 1. VIP produced an acute, dose-dependent (Ki = 3.10(-11) M), 90% inhibition of acetyl-coenzyme A carboxylase activity. These results allow us to elucidate the mechanism of the recently reported inhibitory effect of VIP on glucose oxidation (Vidal, H., Comte, B., Beylot, M., and Riou, J. P. (1988) J. Biol. Chem. 263, 9206-9211) and demonstrate for the first time that balance between fatty acids and glucose as energetic fuels is under neurohormonal control in isolated rat enterocytes.