The sulfhydryl-selective alkylating agent, N-ethylmaleimide (NEM), has been used as a tool to discern whether different binding domains exist on the neuronal serotonin (5-HT) transporter for 5-HT and 5-HT uptake inhibitors (Reith, M. E. A., Allen, D. L., Sershen, H., and Lajtha, A. (1984) J. Neurochem. 43, 249-255; Graham, D., Esnaud, H., Habert, E., and Langer, S. Z. (1989) Biochem. Pharmacol. 38, 3819-3826). However, relatively high concentrations of NEM and long incubation times have been required for inactivation of the transporter-binding site which raises the possibility that NEM is reacting with other nucleophilic groups (Smyth, D. G., Blumenfeld, O. O., and Konigsberg, W. (1964) Biochem. J. 91, 589-595). In the present work, the reactivity and essential nature of sulfhydryl groups associated with substrate/inhibitor binding to the neuronal 5-HT transporter was assessed. [3H]Paroxetine, a potent and selective 5-HT uptake inhibitor, was used to label the 5-HT transporter. The effects of a relatively wide range of sulfhydryl reagents on [3H]paroxetine binding in digitoninsolubilized preparations of rat brain neuronal membranes and the relative abilities of different classes of drugs to protect against NEM-induced inactivation of [3H]paroxetine binding were studied. It was observed that digitonin-solubilized preparations were more sensitive than membrane preparations to the inactivating effects of NEM. The pKa of the reactive group was estimated to be 6.17, in the range expected for a reactive sulfhydryl. Sulfhydryls essential to ligand binding reacted preferentially with hydrophobic compounds (p-hydroxymercuribenzoate = dithiobisnitrobenzoate > methyl methanethiosulfonate > N-phenylmaleimide > N-ethylmaleimide) and were unreactive toward hydrophilic reagents such as iodoacetate and iodoacetamide. 5-HT, 5-HT uptake inhibitors and cocaine protected the digitonin-solubilized transporter from NEM-induced inactivation while the amphetamine-related releasing agents p-chloroamphetamine and fenfluramine were ineffective. The observation that the binding of some, but not all, ligands requires reduced sulfhydryl groups, suggests that differential mechanisms and/or different binding domains do exist for agents which interact at the neuronal 5-HT transporter.