[(11)C]befloxatone is a high-affinity, reversible, and selective radioligand for the in vivo visualization of the monoamine oxidase A (MAO-A) binding sites using positron emission tomography (PET). The multi-injection approach was used to study in baboons the interactions between the MAO-A binding sites and [(11)C]befloxatone. The model included four compartments and seven parameters. The arterial plasma concentration, corrected for metabolites, was used as input function. The experimental protocol-three injections of labeled and/or unlabeled befloxatone-allowed the evaluation of all the model parameters from a single PET experiment. In particular, the brain regional concentrations of the MAO-A binding sites (B'(max)) and the apparent in vivo befloxatone affinity (K(d)) were estimated in vivo for the first time. A high binding site density was found in almost all the brain structures (170+/-39 and 194+/-26 pmol/mL in the frontal cortex and striata, respectively, n=5). The cerebellum presented the lowest binding site density (66+/-13 pmol/mL). Apparent affinity was found to be similar in all structures (K(d)V(R)=6.4+/-1.5 nmol/L). This study is the first PET-based estimation of the B(max) of an enzyme.