We studied the characteristics of [3H]cocaine binding to membranes prepared from whole guinea pig brain. Cocaine binding was specific and saturable. A one-site binding model fit the data adequately: the Kd value of [3H]cocaine was 44 nM with a Bmax value of 280 fmol/mg protein. The rank order of potency for the [3H]cocaine binding site was paroxetine > clomipramine > (-)-cocaine > fluoxetine > mazindol > desipramine > GBR12909 > phencyclidine > benztropine > GBR12935 > (+)-cocaine. The IC50 values of these drugs for inhibition of [3H]cocaine binding were highly correlated with their IC50 values for inhibition of [3H]5-HT uptake into synaptosomes prepared from whole guinea pig brain. High affinity 5-HT uptake inhibitors produced dose-dependent wash-resistant (pseudoirreversible) inhibition of [3H]cocaine binding. The wash-resistant inhibition produced by paroxetine was due to an increase in the Kd of [3H]cocaine binding sites, and was accompanied by an increase in the dissociation rate, consistent with an allosteric mechanism. These studies suggest that, using membranes prepared from whole guinea pig brain, [3H]cocaine labels a binding site associated with serotonin transporter and that paroxetine and cocaine bind to different sites on the serotonin transporter.