Ceramide, the metabolic product of signaling molecule sphingomyelin, has been implicated in cardiac Ca2+ regulation. To study the possible role of ceramide in the pathogenesis of diabetic cardiomyopathy, we examined the effects of ceramide on the cardiac contractility of cultured ventricular myocytes under control and simulated diabetic environments. Adult rat ventricular myocytes were maintained in normal (NG, 5.5 mM) or high glucose (HG, 25.5 mM) medium for 24 hr in the absence or presence of the membrane-permeant ceramide analog C2-ceramide, ceramide glucosyltransferase inhibitor D,L-threo-1-pheny-2-decanoylamino-3-morpholino-1-propanol (PDMP), or the inactive ceramide analog C2-dihydroceramide. Contractile indices analyzed included peak shortening (PS), time-to-PS (TPS), time-to-90% relengthening (TR90), maximal velocity of shortening/relengthening (+/- dL/dt), and intracellular Ca2+ fura-2 fluorescence intensity (FFI). Myocytes maintained in HG medium displayed reduced PS and +/- dL/dt associated with prolonged TR90 and normal TPS compared to NG myocytes. Interestingly, the HG-induced mechanical dysfunctions were significantly attenuated by C2-ceramide or PDMP. C2-ceramide did not affect the myocyte mechanics in NG myocytes although PDMP shortened TPS without affecting any other indices. The HG-induced contractile abnormalities were not altered by inactive ceramide analog C2-dihydroceramide (except +/- dL/dt). Fura-2 fluorescence recording revealed that HG reduced baseline as well as stimulated intracellular Ca2+ levels, which may be abolished by both C2-ceramide and PDMP. These data suggest that alteration of ceramide signaling may play a role in the pathogenesis of diabetic cardiomyopathy.