In ischemic-reperfused myocardium, myocardial cells are jeopardized not only by reoxygenation-induced hypercontracture but also by the development of a transsarcolemmal osmotic gradient. Here the question of whether osmotic fragility of cardiomyocytes can be reduced by interventions during reoxygenation was addressed. Isolated ventricular cardiomyocytes (from adult rats), exposed to 120 min of hypoxia and subsequent reoxygenation, were used as model. With reoxygenation, medium osmolarity was reduced from 270 to 80 mosM. Loss of sarcolemmal integrity was characterized by enzyme loss from cells (creatine kinase and lactate dehydrogenase). Cardiomyocytes reoxygenated after 120 min of hypoxia hypercontracted, but enhanced enzyme loss was observed only at 80 mosM. The nitric oxide (NO) donors 3-morpholinosydnonimine (10 mM), sodium nitroprusside (10 mM), S-nitroso-N-acetyl-DL-penicillamine (100 microM), and the antilipid peroxidant diphenylphenylenediamine (DPPD, 2.5 microM) reduced enzyme loss with hyposmolar reoxygenation. Agents activating guanosine 3',5'-cyclic monophosphate (cGMP)-dependent pathways [atrial natriuretic peptide (1 microM), urodilatin (1 microM), and 8-bromo-cGMP (10 mM)], the contractile inhibitor 2,3-butanedione monoxime (10 mM), and the SIN-1 metabolite SIN-1C (10 mM) did not protect cardiomyocytes against osmotic fragility. The results show that increased osmotic fragility of isolated adult rat cardiomyocytes can be prevented at the time of reoxygenation by NO donors and DPPD in a cGMP-independent way.