1. 1. Rat small intestinal epithelial cells were incubated in vitro at 30°C in bicarbonate buffer, pH 7,4, with 2 mM [1- 14C]pyruvate. The rate of formation of 14CO 2 was inhibited by 5 mM octanoate (58%), 5 nM d,l-3-hydroxybutyrate (37%) and 5 nM acetate (17%). No significant inhibition was observed, when 5 mM propionate was used. 2. 2. On incubation with Mg 2+—ATP, the pyruvate dehydrogenase complex (EC 1.2.4.1) in hobogenates from intestinal epithelial cells was rapidly inactivated. After inactivation, further incubation with 10 nM MgCl 2 and an ATP-removing system resulted in a time-dependent reactivation, suggeting a regulation of the pyruvate dehydrogenase complex in intestine by phosphorylation and dephosphorylation of pyruvate dehydrogenase. 3. 3. Measurement of the pyruvate dehydrogenase complex in extracts from cells, which had been incubated under different conditions, provided evidence that interconversion of the pyruvate dehydrogenase complex determines only to a minor extent the rate of pyruvate oxidation. Intraluminal loading in situ with 5 mM octanoate, compared with 5 nM glucose, gave a slight inactivation of the pyruvate dehydrogenase complex estimated in extracts from freeze-clamped tissue.