We have investigated whether nucleoside drugs that induce or protect neurones against apoptosis might directly activate or inhibit mitochondrial permeability transition (mPT) since opening of the mPT pore can promote release of cytochrome c and apoptosis, while its closure can prevent these changes. We found that the pro-apoptotic pyrimidine analogues cytosine β-d-arabinofuranoside and cytosine β-d-arabinofuranoside 5′-triphosphate, which activated apoptosis in post-mitotic neurones without incorporation into nuclear DNA, induced rapid calcium-dependent mitochondrial swelling of isolated liver mitochondria in a dose-dependent manner. Induction of up to 50 and 80%, respectively, of maximal swelling induced by high calcium was obtained at 1mM concentrations, which also promoted a 17-fold increase in the release of cytochrome c. Both activities were inhibited by cyclosporine A to unstimulated levels; dCTP had no effect. In contrast, the anti-apoptotic adenine analogues, 3-methyladenine (3-MA) and olomoucine (but not iso-olomoucine), inhibited swelling induced by calcium or phenylarsine oxide in a dose-dependent manner at concentrations that protect neurones from apoptosis. Both compounds also inhibited the release of cytochrome c (by 82%, 20mM 3-MA and 95%, 0.9mM olomoucine), similar to the inhibition obtained with cyclosporine A, and 5mM ADP or ATP. Similar inhibitory effects with olomoucine and 3-MA were found in isolated heart mitochondria. These studies identify the mPT as an important target for hitherto untested pro- and anti-apoptotic nucleoside-based drugs and suggest that screening for mPT modulation is an important component in the validation of a drug’s mechanism of action.