The mitochondrial protein cytochrome c has been identified as one of the key signalling molecules of apoptosis. In the present study, we used primary neuronal cultures to investigate whether cytochrome c was released from the mitochondria into the cytosol and subsequently into the culture medium during staurosporine-induced apoptosis and whether extracellular cytochrome c modulates the degree of damage caused by staurosporine. We found the cytochrome c content in the mitochondria decreased 24 h after and increased in the cytosol 8 h after staurosporine was added to the culture medium. The cytochrome c content of the culture medium increased from 8 h up to 24 h after starting the staurosporine treatment. In parallel with the release of cytochrome c into the culture medium, membrane leakage occurred as determined by the release of LDH. Addition of cytochrome c accelerated, whereas the addition of anti-cytochrome c antibodies reduced staurosporine-induced neuronal death suggesting a pro-apoptotic role of cytochrome c released into the culture medium. Under control conditions, extracellularly added cytochrome c (25 ng/ml), which was in the range of the amount of cytochrome c released from staurosporine-treated neurons into the culture medium, increased the percentage of apoptotic neurons to 30% compared with 18% in vehicle-treated controls. Our results suggest that the release of cytochrome c into the extracellular space contributes to neuronal apoptosis induced by staurosporine.