The colocalization of NADPH-diaphorase and acetylcholinesterase activities in the rat main and accessory olfactory bulbs has been studied by successive double histochemical staining of the same sections. In the main olfactory bulb, three patterns of glomerular labeling were found: typical/NADPH-diaphorase-positive, typical/NADPH-diaphorase-negative, and atypical/NADPH-diaphorase-negative glomeruli. Although both enzymatic activities were present in periglomerular cells and superficial short-axon cells, colocalization of NADPH-diaphorase and acetylcholinesterase was not observed in these neuronal types. By contrast, both enzymes were colocalized in a small subpopulation (less than 3% of NADPH-diaphorase- or acetylcholinesterase-positive cells) of short-axon cells located in the external plexiform layer, internal plexiform layer, granule cell layer, and white matter. In the accessory olfactory bulb, deep short-axon cells were the only neurons where both enzymes were present, and colocalization of both markers was observed in some of these cells located in the granule cell layer.