To gain further insight into the mechanism by which irradiation of mitochondria in the presence of haematoporphyrin derivative (Photofrin II) (PF II) causes impairment of mitochondrial oxidative phosphorylation, the rate of ADP/ATP exchange via the ADP/ATP translocator was measured fluorometrically is isolated rat liver mitochondria. In accord with noncompetitive inhibition, PF II photosensitization decreases the maximum rate of exchange Vmax (20.8 and 9.6 nmol ATP effluxed min-1 x mg protein in the control and after 2 min irradiation, respectively) without changing the ADP affinity for the carrier (Km = 5 microM in both cases). Comparison of the rate of oxygen uptake by mitochondria stimulated by either ADP or by the uncoupler carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) confirms that the adenine nucleotide carrier is a major target of photodynamic action which causes oxidative phosphorylation impairment.