Phenotypic diversity underlies the complex functioning of the nervous system. One characteristic in which neurons differ from one another is the kind of molecules that they use for intercellular signalling. The classical neurotransmitter acetylcholine, synthesized by the enzyme choline acetyltransferase, is used by five groups of neurons in the rat spinal cord. Another messenger is nitric oxide, which is synthesized by nitric oxide synthase. Neurons that express nitric oxide synthase can be stained specifically by NADPH diaphorase histochemistry. In the spinal cord, approximately five groups of neurons are labeled by the diaphorase reaction, and some of these populations overlap with cholinergic groups. To determine the proportions of neurons that co-express choline acetyltransferase and nitric oxide synthase, we performed choline acetyltransferase immunocytochemistry and diaphorase histochemistry on single sections of rat spinal cord. Some cell types were single-labeled: somatic motor neurons were choline acetyltransferase-immunoreactive only, and neurons in lamina II were diaphorase-positive only. Four cell groups included double-labeled cells. Autonomic motor neurons were either double-labeled (62%) or choline acetyltransferase-only (37%), partition cells in lamina VII were double-labeled (54%) or choline acetyltransferase-only (45%), neurons in laminae III-V of the dorsal horn were double-labeled (70%) or diaphorase-only (27%), and neurons surrounding the central canal were double-labeled (56%), choline acetyltransferase-only (23%) or diaphorase-only (21%). These data indicate that certain spinal cord populations may be heterogeneous with regard to the intercellular messenger phenotypes involving acetylcholine and nitric oxide.(ABSTRACT TRUNCATED AT 250 WORDS)