Cortical function has been suggested to be highly compromised by repeated heroin self-administration. We have previously shown that street heroin induces apoptosis in neuronal-like PC12 cells. Thus, we analysed the apoptotic pathways involved in street heroin neurotoxicity using primary cultures of rat cortical neurons. Our street heroin sample was shown to be mainly composed by heroin, 6-monoacetylmorphine and morphine. Exposure of cortical neurons to street heroin induced a slight decrease in metabolic viability, without loss of neuronal integrity. Early activation of caspases involved in the mitochondrial apoptotic pathway was observed, culminating in caspase 3 activation, Poly-ADP Ribose Polymerase (PARP) cleavage and DNA fragmentation. Apoptotic morphology was completely prevented by the non-selective caspase inhibitor z-VAD-fmk, indicating an important role for caspases in neurodegeneration induced by street heroin. Ionotropic glutamate receptors, opioid receptors and oxidative stress were not involved in caspase 3 activation. Interestingly, street heroin cytotoxicity was shown to be independent of a functional mitochondrial respiratory chain, as determined using NT-2 rho(0) cells. Nonetheless, in street heroin-treated cortical neurons, cytochrome c was released, accompanied by a decrease in mitochondrial potential and Bcl-2/Bax. Pure heroin hydrochloride similarly decreased metabolic viability but only slightly activated caspase 3. Altogether, our data suggest an important role for mitochondria in mediating street heroin neurotoxic effects.