The novel gaseous neuromediator nitric oxide is thought to play an important role in development and plasticity. 20 Despite this, gene-knockout mice lacking neuronal (Type I) nitric oxide synthase exhibit relatively normal brain development and behavior. The nervous system of these mice (especially the forebrain) retains some calcium-dependent nitric oxide synthesis, presumably reflecting other isozymes. 8 Type I nitric oxide synthase has NADPH-dependent diaphorase activity. 2,3,7,16 However, this stain also recognizes other isozymes, 18,23 and it remains controversial whether all diaphorase-positive neurons contain Type I nitric oxide synthase. 13,22 To assess whether neurons containing another isoform of nitric oxide synthase may be present in the forebrain of normal rodents, we studied co-localization of diaphorase staining with immunocytochemistry for Type I nitric oxide synthase. Co-localization was complete in the striatum, but some neurons deep in cortex were diaphorase-positive and immunonegative, and therefore may contain a splice variant 15 or novel isozyme of nitric oxide synthase.