Control of coenzyme A (CoA) synthesis was studied in isolated perfused rat hearts. The data obtained support the hypothesis that phosphorylation of pantothenic acid by pantothenate kinase is the flux-generating reaction in the pathway of CoA synthesis. This reaction operated in the cell far removed from its thermodynamic equilibrium; it was saturated with substrates under all conditions studied; and the concentration of substrate changed in the opposite direction to flux when flux was altered. The reaction was subject to control by external factors associated with oxidation of glucose, pyruvate, or palmitate. CoA synthesis from 4'-phosphopantothenic acid was not inhibited by glucose and pyruvate, suggesting that pantothenate kinase is the only reaction in the pathway that is controlled in isolated hearts. Maximum rates of CoA synthesis in perfused hearts with pantothenate kinase stimulation were dependent on a supply of exogenous cysteine. Perfusate [14C]cysteine was incorporated into intermediates of this pathway and CoA. When protected from oxidation to cystine by low concentrations of dithiothreitol, 0.1 mM cysteine in the perfusate resulted in maximum rates of CoA synthesis. Evidence was obtained that indicates that addition of cysteine relieves a substrate limitation at the 4'-phosphopantothenyl cysteine synthase reaction.