Objectives: Anthracyclines cause apoptotic death in many cell types through activation of the ceramide pathway. We tested the hypothesis that doxorubicin induces cardiac myocyte apoptosis through ceramide generation. Methods: Adult rat ventricular myocytes were grown in the presence of 10% fetal calf serum, and exposed to 0.5 μM doxorubicin (Dox) for 1 h on the day of cell isolation (day 0). We used the membrane-permeant ceramide analog C₂-ceramide (C₂-cer) to mimic the effects of endogenous ceramide and PDMP to induce endogenous ceramide accumulation. Apoptosis was assessed upon morphological criteria and DNA fragmentation by the TUNEL method and agarose gel electrophoresis. Ceramide concentration was assessed using the DAG kinase assay. Results: Myocyte exposure to Dox was associated with cellular and nuclear alterations typical of apoptosis on day 7 but not on day 3. At day 7, the percentage of TUNEL-positive myocytes was markedly increased in Dox-treated cultures compared to control (Cl) cultures (82±3 vs. 12±1%, n=7; p<0.001); internucleosomal DNA fragmentation was confirmed by the observation of DNA ladders. These alterations were associated with an increase in the intracellular ceramide concentration (1715±243 vs. 785±99 pmol/mg prot, n=5; p<0.01), a phenomenon also detected on day 3 (731±59 vs. 259±37 pmol/mg prot, n=5; p<0.001). Incubation of myocytes at day 0 with 50 μM C₂-cer induced rapid cell shrinkage and DNA fragmentation (45±3 vs. 10±1% TUNEL-positive myocytes on day 1 in C₂-cer-treated and Cl cultures, respectively; n=6, p<0.001). Myocyte exposure to 10 μM PDMP for 7 days (n=5), caused ceramide accumulation (1.7-fold increase vs. Cl, p<0.01), and a marked increase in the percentage of TUNEL-positive myocytes (62±6 vs. 11±3% in Cl cultures, p<0.001). Ventricles of rats injected intraperitoneally with a cumulative dose of 14 mg/kg Dox over a period of 2 weeks also showed an increased ceramide concentration 2 weeks later (11.01±0.64 vs. 5.24±0.88 pmol/mg prot, n=6; p<0.001). Conclusion: Our study confirms the existence of a functional ceramide pathway related to apoptosis in cardiac myocytes, and points to its possible involvement in doxorubicin-induced cardiomyopathy.