Somatostatin, neuropeptide Y, and nicotinamide adenine dinucleotide phosphate-diaphorase are colocalized within a small population of medium aspiny neurons in the caudate-putamen of the rat. The extent of colocalization, however, appears to be in dispute. In order to examine the question of colocalization between these three neuroactive substances, a series of double-labelling experiments was performed. This was accomplished by combining immunocytochemistry for somatostatin or neuropeptide Y or enzyme histochemistry for nicotinamide adenine dinucleotide phosphate-diaphorase with in situ hybridization for somatostatin and/or neuropeptide Y mRNA. The results of such analysis indicate that nicotinamide adenine dinucleotide phosphate-diaphorase and somatostatin mRNA are 100% colocalized throughout the caudate-putamen, except for the area bordering the globus pallidus. All neurons that contain neuropeptide Y contain somatostatin message. Only 84% of the neurons that contain somatostatin mRNA, however, also contain neuropeptide Y. Neurons that contain somatostatin 28 but not neuropeptide Y are found throughout the caudate-putamen. These results indicate that the somatostatin neuron population in the rat caudate-putamen is not homogeneous. Instead, the medium aspiny neuron population is actually composed of several subpopulations based on the content of neuroactive substances.