Tritiated sertraline, a radiolabeled form of a potent and selective inhibitor of serotonin uptake, was found to bind with high affinity to rat whole brain membranes. Characterization studies showed that [3H] sertraline binding occurred at a single site with the following parameters: KD 0.57 nM, Bmax 821 fmol/mg protein, nH 1.06. This binding was reversible; the dissociation constant calculated from kinetic measurements (KD 0.81 nM) agreed with that determined by saturation binding experiments. [3H] Sertraline binding in the presence of serotonin, paroxetine, fluoxetine or imipramine suggested competitive inhibition of binding (large increase in KD with little change in Bmax). The rank order of potency of inhibition of [3H] sertraline binding was similar to that of inhibition of serotonin uptake for known uptake inhibitors and the 1-amino-4-phenyltetralin uptake blockers. A marked decrease in ex vivo [3H] sertraline binding in the brain of rats 7 days after treatment with p-chloroamphetamine was consistent with the loss of serotonin uptake sites induced by this agent. The results of our study indicated that [3H] sertraline labels serotonin uptake sites in rat brain.