Various studies performed on different models have demonstrated that apoptosis occurs in ischaemic-reperfused myocardium in vivo; however, the individual contribution of ischaemia and reperfusion to CMC (cardiomyocyte) apoptosis remains uncertain. We have determined the main inducer of CMC apoptosis in ischaemia-reperfusion by exposing CMCs to either 30 min ischaemia followed by reperfusion or to 25-OH-cholesterol (25-hydroxycholesterol) for 1–3 days. Both ischaemia-reperfusion and exogenous oxidants increased the Bax/Bcl-2 ratio, a favourable effect for the apoptotic process. However, apoptosis was not observed in ischaemic CMCs in the absence of reperfusion. Moreover, reperfusion after 30 min ischaemia did not make an important contribution to CMC apoptosis in culture in terms of caspase 3 activation. In contrast, 25-OH-cholesterol promoted CMC apoptosis by a caspase 3-dependent mechanism that involved the transcriptional activation of the pro-apoptotic protein, Bax and post-translational degradation of the anti-apoptotic protein, Bcl-2. From these results, we conclude that CMC apoptosis is not induced by ischaemia per se, but by the oxidants from the surrounding environment at the time of reperfusion. These exogenous oxidants exacerbate the alterations induced by ischaemia and complete the apoptotic process at the time when ATP and glucose levels are restored.