Increasing therapeutic use is made of purines for the treatment of ischemic heart disease, but little is known about regulatory mechanisms involved. Therefore we perfused isolated rat hearts with 0.02 mmol/l [8-14C]hypoxanthine or inosine. Under normoxic conditions about 1% is taken up by the heart and partially used for synthesis of ATP and GTP at a rate of 0.4 and 0.1 nmol X min-1 X g dry wt-1, respectively. After relatively mild ischemia (coronary flow reduction of 70% for 20 min), no increase in myocardial purine uptake is observed, but ATP and GTP synthesis rates are doubled (P less than 0.001). D-Ribose stimulates the hypoxanthine incorporation rate in normoxic perfused rat hearts to 1.1 and 0.5 nmol X min-1 X g dry wt-1 for ATP and GTP, respectively, which is further increased during postischemic perfusion. About 80% of the [8-14C]inosine or [8-14C]hypoxanthine passes through the heart unchanged, while 15% is converted to (hypo)xanthine and uric acid. We conclude from these experiments that inosine and hypoxanthine incorporation into ATP and GTP is at least partly regulated by the availability of 5-phosphoribosyl-1-pyrophosphate.