Nitrogen monoxide (NO) has important cardiovascular actions, and it has been suggested that they may be partly mediated by the reaction with protein sulfhydryl groups to produce S-nitrosothiols. In this work we describe and test a method that allows S-nitrosothiol detection in crude membrane preparations obtained from isolated perfused rat hearts. Isolated rat hearts were perfused under control conditions or in the presence of the NO donors SIN-1 and isosorbide dinitrate. Additional hearts were subjected to 10-20 min of ischemia followed or not by 10-20 min of reperfusion. At the end of perfusion a crude membrane fraction was prepared, and S-nitrosothiol concentration was assayed fluorometrically, on the basis of 2,3-naphthotriazole production from 2,3-diaminonaphthylene. The sensitivity of the method, as evaluated using S-nitrosoalbumin, was on the order of 1-2 pmol/mg of protein. S-nitrosothiols were undetectable under control conditions, as well as after ischemia or ischemia-reperfusion. On the other hand, significant S-nitrosothiol formation was observed after infusion of SIN-1 or isosorbide dinitrate (26.4 +/- 7.4 and 19.9 +/- 5.6 pmol per mg of protein, respectively). In conclusion, S-nitrosothiol production was observed in rat heart membranes after exposure to NO donors, while S-nitrosothiol concentration was below the sensitivity limits of the assay either under baseline conditions or after acute ischemia and reperfusion.