We report here saturation analysis of [3H]cocaine binding in various mouse brain regions, and the necessary structure-activity relationships for cocaine congeners to inhibit Na+-dependent [3H]cocaine binding and [3H]dopamine uptake in the mouse striatum, and to inhibit [3H]cocaine binding that cannot be stimulated by Na+ and [3H]serotonin uptake in the mouse cerebral cortex. Generally similar structure-activity relationships were noted for all these processes. The ester linkage between the tropane and phenyl rings was not required for activity, in contrast to the configuration of the groups on C2, and to a lesser extent C3, in the tropane ring. Stereospecificity was evident from the differences between cocaine and (+)-pseudococaine, and between WIN 35,065-2 and WIN 35,065-3. There were remarkable differences between the above structure-activity relationships and those for local anesthetic activity of cocaine congeners, indicating that sodium channels were not labeled to a measurable extent with [3H]cocaine under the present conditions. Preliminary data indicated a significant correlation between the potencies of cocaine congeners in inhibiting the Na+-dependent binding of [3H]cocaine and their potencies in inducing stereotyped sniffing upon intraventricular administration.