We have examined the populations of neurons in the neostriatum of both rat and cat that are immunoreactive for glutamate decarboxylase, [Leu]enkephalin, [Met]enkephalin and substance P. Neurons that were immunoreactive for glutamate decarboxylase made up 47% of the neurons in our samples from the rat and ranged from 39 to 49% of the neurons in the cat. Those immunoreactive for [Leu]enkephalin made up 44-49% of the neurons in rat neostriatum, and 38-47% in the cat, and those immunoreactive for [Met]enkephalin made up 36-41% of the neurons in rat and 43-49% of the neurons in the cat. Substance P-immunoreactive neurons made up 30-38% of neurons in rat and 32-39% in cat. Most substance P neurons (particularly the most darkly staining ones) were, however, clustered such that they were most numerous in the patch compartment of neostriatum; within the patches the substance P neurons comprised 59% of neurons in the rat and 55% in cat, but in the matrix substance P neurons comprised only 32% of neurons in the rat and 25% in the cat. Samples taken from sections processed for two-color double labeling immunocytochemistry revealed that 12% of neurons label for both glutamate decarboxylase and [Leu]enkephalin, 12% for both glutamate decarboxylase and [Met]enkephalin, 11-12% for both glutamate decarboxylase and substance P, and 17% for both [Met]enkephalin and substance P. These results provide evidence for chemical heterogeneity within the medium-sized neostriatal neurons, and provide the first evidence for coexistence of glutamate decarboxylase and substance P within a single neuron, and the first evidence for the coexistence for substance P and [Met]enkephalin within single neurons of the central nervous system.