Inosine was administered as a continuous intravenous infusion (100 mg/kg per hr) in rats treated with isoproterenol (25 mg/kg, s.c.). After 24 hr, the isoproterenol-induced decline in myocardial ATP and total adenine nucleotides was attenuated by inosine. Infusion of ribose (100 mg/kg per hr) had a similar cardioprotective influence. The effect of inosine may have been due to the incorporation into cardiac adenine nucleotides of the degradation products of inosine, either of the ribose moiety via the de novo synthesis pathway or of hypoxanthine via the salvage pathway utilizing 5-phosphoribosyl-1-pyrophosphate. To decide this, [8-(14)C]inosine was injected intravenously 4 hr after administration of isoproterenol. Its incorporation into myocardial adenine nucleotides was enhanced. Furthermore, the effect of inosine on the de novo synthesis of adenine nucleotides in normal and isoproterenol-stimulated hearts was studied and compared to that of ribose. The normal rate was attenuated and the isoproterenol-elicited increase was reduced by inosine. In contrast, ribose stimulated adenine nucleotide biosynthesis in the control rat heart and potentiated the isoproterenol-induced enhancement. These results indicate that the effect of inosine is not due to the metabolism of ribose-1-phosphate via the pentose phosphate and the de novo synthesis pathways, but rather to the 5-phosphoribosyl-1-pyrophosphate-dependent salvage of hypoxanthine. Obviously, this pathway is of such a magnitude that the isoproterenol-evoked decline in myocardial adenine nucleotide levels is attenuated.