Control of coenzyme A synthesis was studied in isolated, perfused rat hearts. Pantothenic acid (PA), coenzyme A, and intermediates in the the pathway were separated by high pressure liquid chromatography. The amount of 14C label in each of the metabolites was determined in tissue extracts when [14C]PA was supplied in the perfusate. The rate-controlling steps in the pathway were determined by measuring the net rate of [14C]PA flux through each of the reactions. The data indicated that the primary site of control in the pathway was the pantothenate kinase-catalyzed reaction, the first intracellular step in the conversion of PA to CoA. The rate of this reaction was inhibited by including glucose, pyruvate, fatty acids, or beta-hydroxybutyrate in the perfusate of isolated hearts. Pyruvate and beta-hydroxybutyrate caused a much greater inhibition than did glucose. Insulin was a strong inhibitor, but only in the presence of glucose. Insulin had no effect in hearts receiving either no substrate or palmitate as substrate. Collectively, these data indicated that an unknown tissue metabolite whose level changed with each of these substrates and insulin is a strong regulator of pantothenate kinase. Synthesis of CoA occurred in both the cytosolic and mitochondrial compartments. Accelerated mitochondrial CoA synthesis appeared to be dependent upon the production and accumulation of 4'-phosphopantotheine, which occurred only when pantothenate kinase was stimulated.