The in vitro and In vivo effects of fluoxetine (and its active metabolite norfluoxetine) on mitochondrial respiration and F₀F₁-ATPase were studied, respectively, in mitochondria and submitochondrial particles isolated from rat liver. Fluoxetine in vitro inhibited state 3 mitochondrial respiration for α-ketoglutarate and succinate oxidations (50% of effect at 0.25 and 0.35 mM drug concentrations, respectively); stimulated state 4 for succinate; and induced a decrease in the respiratory control ratio (RCR) for both oxidizable substrates. The F₀F₁-ATPase activity was determined at various pH levels in the absence and presence of Triton X-100. The solubilized form was not affected markedly, but an inhibition, apparently non-competitive, was observed for the membrane-bound enzyme, with 50% of the effect at a 0.06 mM drug concentration in pH7.4. These results suggest that fluoxetine in vitro acts on F₀F₁ATPase through direct interaction with the membrane F₀ component (similar to oligomycin), or first with mitochondrial membrane and then affecting F₀. A very similar behavior concerning the respiratory parameters and F₀F₁-ATPase properties was observed with norfluoxetine. The in vivo studies with fluoxetine showed stimulation of mitochondrial respiration in state 4 for α-ketoglutarate or succinate oxidations in acute or prolonged treatments (1 hr after a single i.p. dose of 20 mg of drug/kg of body weight, and 22 hr after 12 days of treatment with a daily dose of 10 mg/kg of body weight, respectively), indicating uncoupling of oxidative phosphorylation. Pronounced changes were not observed in the K_0.5 values of F₀F₁-ATPase catalytic sites, but the V_max decreased during the prolonged treatment. The results show that fluoxetine (as well as norfluoxetine) has multiple effects on the energy metabolism of rat liver mitochondria, being potentially toxic in high doses. The drug effects seem to be a consequence of the drug and/or metabolite solubilization in the inner membrane of the mitochondria.