The effects of tacrolimus (FK 506) on brain phosphorylation have been investigated in vitro using mitochondria isolated from rat brain. Respiratory control ratio (RCR), oxygen consumption, ATP synthesis and enzymatic activities of involved complexes have been measured to assess the mechanisms of action of tacrolimus. Our data show that this drug decreases RCR and ATP synthesis. This effect is quantitatively limited after a single application of the drug (14%), concentration-dependent and biphasic, the respective effect 50%-concentration (EC₅₀) being 0.129 and 247 nM, each step corresponding to 50% of the total oxygen consumption inhibition. Tacrolimus acts mainly as an inhibitor of ubiquinol-cytochrome c reductase (complex III), competing at least partly with antimycin A or myxothiazol, the corresponding EC₅₀ being 0.27 and 103 nM respectively. Tacrolimus inhibits also complex V i.e. ATPase activity (40%) and ATP synthase activity (30%) in a concentration-dependent manner, the relevant EC₅₀ being 78 and 394 nM respectively. These data may be relevant for the protective effect of tacrolimus observed in ischemia-reperfusion, which may be due to its inhibition of both complex III, where Reactive Oxygen Species (ROS) are generated, and complex V, where ATP is depleted by ATPase activation. It may also be related to neurotoxicity occurring along chronic administration of tacrolimus in humans.