The effects of inosine (INO) on substrate metabolism and rigor formation in ischemic myocardium were examined in isolated rabbit hearts. Metabolite content was assessed in tissue extracts by chemical analysis and in the whole heart by 13C and 31P nuclear magnetic resonance spectroscopy. In ischemic hearts metabolizing either [3-13C]pyruvate or [1-13C]glucose, 1 mM INO increased both total and 13C-labeled alanine content; lactate content was unaffected. At 3 minutes of ischemia, tissue alanine was 1.81 +/- 0.11 microM/g wet wt (mean +/- SEM) in hearts perfused with pyruvate+INO versus 1.23 +/- 0.15 microM/g wet wt in hearts perfused with pyruvate alone (p less than 0.05). INO reduced tissue glycogen during ischemia in pyruvate-perfused hearts. Tissue alanine content in ischemic hearts that were supplied glucose+INO (1.29 +/- 0.13 microM/g wet wt) was greater than in ischemic hearts supplied glucose alone (0.65 +/- 0.14 microM/g wet wt). Alanine was found to originate from pyruvate and was a glycolytic end product in glucose-perfused hearts. INO raised the [3-13C]alanine/[3-13C]lactate ratio in ischemic, intact hearts (glucose = 0.24 +/- 0.07 versus glucose+INO = 0.60 +/- 0.09; pyruvate = 0.49 +/- 0.08 versus pyruvate+INO = 0.89 +/- 0.08). At 7 minutes of ischemia, ATP content fell to 70 +/- 3% with glucose+INO versus 58 +/- 5% with glucose alone. Rigor (stone heart) was delayed from 14.7 +/- 1.3 to 23.2 +/- 1.6 minutes with INO. INO did not change ATP content in ischemic hearts that were supplied pyruvate but delayed rigor (pyruvate = 9.9 +/- 1.2 minutes; pyruvate+INO = 15.6 +/- 1.0 minutes), possibly at the expense of glycogen. Supplemental glucose improved the effectiveness of INO with pyruvate to preserve ATP (pyruvate+glucose = 42 +/- 6%; pyruvate+glucose+INO = 72 +/- 6%) and further delayed rigor (pyruvate+glucose = 13.3 +/- 1.5 minutes; pyruvate+glucose+INO = 20.3 +/- 1.8 minutes). Glucose metabolism supported improved energetic and contractile states in ischemic hearts treated with INO. Thus, cardioprotection of the ischemic heart by INO was associated with preservation of functional integrity and improved energy production due to increased glycolytic activity. Activation of glycolysis in the presence of INO was accommodated by augmented alanine production without the additional accumulation of lactate.