Pyruvate dehydrogenase activity was measured in extracts of isolated kidney cortex tubules, prepared by collagenase treatment. The measured activities were comparable to those described previously for whole kidney homogenates.Incubation of tubules in vitro in the absence of exogenous substrates led to a steady increase in enzyme activity over 30 min. This increase was not significantly changed by the addition of several gluconeogenic substrates including lactate, glutamine, glutamate, glycerol, fructose and malate. 1 mM oleate, 5 mM acetate and 20 mM 2-oxoglutarate however, prevented this increase completely. I n contrast, glucose and dihydroxyacetone accelerated the activation of pyruvate dehydrogenase during incubation in vitro. 20 mM pyruvate very rapidly activated pyruvate dehydrogenase with a following decrease below the activity of control experiments without substrate. 1 mM oleate and 20 mM 2-oxoglutarate counteracted the increase in pyruvate dehydrogenase activity caused by glucose and dihydroxyacetone and had no effect in the presence of 20 mM pyruvate.Determination of the inactive part of pyruvate dehydrogenase after addition of purified pyruvate dehydrogenase phosphatase revealed that all changes observed were probably caused by enzymatic interconversion of pyruvate dehydrogenase.The results are compared with previously published observations on the regulationlof pyruvate dehydrogenase in vivo and confirm the role of fatty acids in the regulation of kidney ,cortex pyruvate metabolism. Enzymes. Pyruvate dehydrogenase (EC 1.2.4.1) ; Arylamine acetyltransferase (EC 2.3.1.5). and kidney cortex [5,7] the mechanism by which fatty acids depressed pyruvate oxidation was a matter of speculation. Inhibition of pyruvate dehydrogenase by increased levels of acetyl-CoA [4] and an increase in reduced nicotinamide-adenine dinucleotide have been discussed as possible metabolic mediators of the action of fatty acids. Although these mechanisms have been shown to operate in isolated mitochondria [S] and with purified enzyme preparations [9], their physiological importance was hard to prove in whole cells since intracellular compartmentalization of metabolites and cofactors prevented $ a n exact interpretation of measured overall levels.The