In order to evaluate the effect of one of the main oxysterols derived from cholesterol oxidation, cholesterol-5 alpha,6 alpha-epoxide (epox), on cardiac cells, we have supplemented the culture medium of neonatal rat cardiomyocytes with scalar concentrations of epox (0.1-100 microM). While 0.1 microM epox supplementation was ineffective, epox supplementation in the range 1-100 microM determined a reduction in cellular protein level, without affecting cell viability, and a dose-dependent epox incorporation into cardiomyocyte lipids. Furthermore, in the same concentration range of epox supplementation, a gas chromatographic peak unambiguously identified by gas chromatography-mass spectrometry as cholestane-3 beta,5 alpha,6 beta-triol, an hydrolytic metabolite of epox, was detected. The mechanism of cytotoxicity of epox to cardiomyocytes could be due to the insertion of epox itself into cellular lipids, and to its metabolization to the more toxic triol.