The mitochondrial permeability transition pore (PTP) and associated release of cytochrome c are thought to be important in the apoptotic process. Nitric oxide (NO·) has been reported to inhibit apoptosis by acting on a variety of extra-mitochondrial targets. The relationship between cytochrome c release and PTP opening, and the effects of NO· are not clearly established. Nitric oxide, S-nitrosothiols and peroxynitrite are reported to variously inhibit or promote PTP opening. In this study the effects of NO· on the PTP were characterized by exposing isolated rat liver mitochondria to physiological and pathological rates of NO· released from NONOate NO· donors. Nitric oxide reversibly inhibited PTP opening with an IC50 of 11 nM NO·/s, which can be readily achieved in vivo by NO· synthases. The mechanism involved mitochondrial membrane depolarization and inhibition of Ca2+ accumulation. At supraphysiological release rates (>2 μM/s) NO· accelerated PTP opening. Substantial cytochrome c release occurred with only a 20% change in mitochondrial swelling, was an early event in the PTP, and was also inhibited by NO·. Furthermore, NO· exposure resulted in significantly lower cytochrome c release for the same degree of PTP opening. It is proposed that this pathway represents an additional mechanism underlying the antiapoptotic effects of NO·.