Autoradiographic techniques revealed that 5-hydroxytryptamine1A, 5-hydroxytryptamine1B and 5-hydroxytryptamine1C/2 binding sites are differentially distributed in the spinal cords of adult male rats. In the dorsal horn, 5-hydroxytryptamine1A sites were dense in all laminae; 5-hydroxytryptamine1B sites were more dense in laminae I, III and IV than in lamina II; while 5-hydroxytryptamine1C/2 sites were very sparse. The dorsal commissure gray matter also exhibited very dense 5-hydroxytryptamine1A and 5-hydroxytryptamine1B binding. In the intermediate and central gray matter, all three sites were moderately dense at autonomic levels, with exceptionally dense1C/2 binding restricted to the intermediolateral nucleus at rostral thoracic levels. In the ventral horn, 5-hydroxytryptamine1A and 5-hydroxytryptamine1B sites were very sparse (except for very dense 5-hydroxytryptamine1A sites located in the dorsolateral nucleus of the pudendal nerve), while 5-hydroxytryptamine1C/2 sites were relatively dense in motor nuclei. Surprisingly, 5-hydroxytryptamine1B sites were moderately dense in the dorsal column corticospinal tract. These studies will provide an anatomical perspective for interpretation of the complex role of 5-hydroxytryptamine in regulating spinal cord function.