The aim of the present studies was to characterise cell death following inhibition of mitochondrial complex I with rotenone in a transformed cell line (RGC-5 cells) and to examine the neuroprotective properties of the flavonoids genistein, epigallocatechin gallate (EGCG), epicatechin (EC) and baicalin. Rotenone-induced cell death of RGC-5 cells results in a generation of reactive oxygen species, a breakdown of DNA, the translocation of membrane phosphatidylserine, an up-regulation of haemoxygenase-1 and is unaffected by necrostatin-1 (inhibitor of necroptosis), z-VAD-fmk (pan caspase inhibitor) or NU1025 (PARP inhibitor) but attenuated with SP600125 (JNK inhibitor). Rotenone-induced toxicity of RGC-5 cells also caused an activation of mitogen-activated kinases indicated by an up-regulation and translocation into mitochondria of p-c-Jun, pJNK and pp38. Exposure of RGC-5 cells to rotenone does not affect apoptosis inducing factor or significantly stimulate caspase-3 activity. EGCG and EC both significantly blunt rotenone toxicity of RGC-5 cells at concentrations of 50 μM while genistein and baicalin were without effect. Significantly, genistein is approximately 20 times less efficacious than EGCG (IC(50) 2.5 μM) and EC (IC(50) 1.5 μM) at inhibiting sodium nitroprusside-induced lipid peroxidation. These studies show that rotenone toxicity of RGC-5 cells is neither necroptosis nor caspase-dependent apoptosis but involves the activation of mitogen-activated kinases and is inhibited by a JNK inhibitor, EGCG and EC. Genistein attenuates lipid peroxidation less efficaciously than EC and EGCG and does not affect rotenone toxicity of RGC-5 cells.