[3H](+)-Pentazocine binding has been characterized in the rat brain. It binds to a single population of binding sites with affinity of about 7 nM and density of 280 fmol/mg protein. [3H](+)-Pentazocine binding is not enriched in the crude synaptic membrane, being about 1/6 of what we found in the crude membrane preparation. The binding, like that for other sigma ligands, was enriched in the microsomal and nuclear fractions. The inhibition by haloperidol, proadifen and d-fenfluramine was the same in the crude synaptic membrane, nuclear and microsomal fractions, suggesting that [3H](+)-pentazocine binds to a homogeneous protein in the different subcellular fractions. Our pharmacological characterization using 45 different drugs suggests that the [3H](+)-pentazocine binding site in rat brain differs from other sigma ligands, like N-propyl-3-(3-hydroxyphenyl)piperidine ([3H](+)-3PPP), N,N'-di(o-tolyl)guanidine ([3H]DTG) and (+)-N-allylnormetazocine ([3H](+)-SKF10,047). [3H](+)-Pentazocine binding in rat brain is inhibited by sigma compounds and some cytochrome P450 ligands, like proadifen and 1-[2-[bis(4-fluoro-phenyl) methoxy]ethyl]-4-[3-phenylpropyl] piperazine (GBR 12909), although with considerably lower potency than reported for other sigma ligands. Other inhibitors are some serotonin uptake blockers or their metabolites and phenylalkylamines.