Lipid droplets (LD) consist of accumulations of triacylglycerols and have been proposed to be markers of ischaemic but viable tissue. Previous studies have described the presence of LD in myocardium surviving an acute coronary occlusion. We investigated whether LD may be protective against cell death secondary to ischaemia-reperfusion injury. The addition of oleate-bovine serum albumin complex to freshly isolated adult rat cardiomyocytes or to HL-1 cells resulted in the accumulation of intracellular LD detectable by fluorescence microscopy, flow cytometry and (1)H-nuclear magnetic resonance spectroscopy. Simulated ischaemia-reperfusion of HL-1 cells (respiratory inhibition at pH 6.4 followed by 30 min of reperfusion) resulted in significant cell death (29.7+/-2.6% of total lactate dehydrogenase release). However, cell death was significantly attenuated in cells containing LD (40% reduction in LDH release compared with control cells, P=0.02). The magnitude of LD accumulation was inversely correlated (r(2)=0.68, P=0.0003) with cell death. The protection associated with intracellular LD was not a direct effect of the fatty acids used to induce their formation, because oleate added 30 min before ischaemia, during ischaemia or during reperfusion did not form LD and did not protect against cell death. Increasing the concentration of free oleate during reperfusion progressively decreased the protection afforded by LD. HL-1 cells labelled with fluo-4, a Ca(2+)-sensitive fluorochrome, fluorescence within LD areas increased more throughout simulated ischaemia and reperfusion than in the cytosolic LD-free areas of the same cells. As a consequence, cells with LD showed less cytosolic Ca(2+) overload than control cells. These results suggest that LD exert a protective effect during ischaemia-reperfusion by sequestering free fatty acids and Ca(2+).