Binding of the radiolabeled active isomer of the neuroleptic sulpiride, [3H]-S-sulpiride, to rat and rabbit striatal membranes was characterized. Regardless of whether the specific binding of [3H]-S-sulpiride was defined with spiperone or the active isomers of butaclamol or flupenthixol, a single homogeneous++ population of binding sites (rat: Kd = 5.6 nM, maximum binding = 590 fmol/mg of protein; rabbit: Kd = 8.3 nM, maximum binding = 540 fmol/mg of protein) was detected. The pharmacological profile of these sites was characteristic of that described for the dopaminergic D-2 receptor subtype. To determine whether [3H]-S-sulpiride and [3H]spiperone label common sites in the striatum, the binding of these two radioligands was compared under similar assay conditions. When specific binding of [3H]spiperone was defined with S-sulpiride, [3H]spiperone labeled the same number of binding sites as [3H]-S-sulpiride despite the fact that the affinity of the sites for [3H]spiperone was 80- to 90-fold higher than for [3H]-S-sulpiride. When specific binding of [3H]spiperone was defined with either (+)-butaclamol or (alpha)-flupenthixol, however, approximately 30% more sites were labeled. The predominant site labeled by [3H]spiperone also possessed the characteristics of the D-2 receptor. It is concluded that [3H]-S-sulpiride under the conditions used is a selective radioligand with which dopamine receptors of the D-2 subtype can be directly measured and localized. [3H]Spiperone can be used to detect the same sites only if specific binding is defined with S-sulpiride.