The developmental expression and cellular distribution of nitric oxide synthase was investigated in the murine cerebellum and in cerebellar neurons developing under controlled in vitro conditions. Cerebellar granule cells expressed nitric oxide synthase only after migration to the internal granule cell layer. Initially, the nascent internal granule cell layer throughout the cerebellum stained uniformly for nitric oxide synthase, but during the second postnatal week, a pattern emerged consisting of clusters of heavily stained granule cells separated by areas of unstained granule cells. This pattern persisted into adulthood. There was a close temporal correlation between innervation of the granule cell layer by mossy fibers and the emergence of granule cell compartments as defined by levels of nitric oxide synthase expression. Granule cells in dissociated cultures derived from cerebellar anlagen prior to mossy fiber innervation also express nitric oxide synthase. The time-course of nitric oxide expression was independent of electrical activity of the neuronal network forming in vitro. However, suppression of spontaneous electrical activity resulted in enhanced nitric oxide synthase expression. These findings indicate that granule cell precursors are endowed with an intrinsic program which regulates nitric oxide synthase induction and which is executed independently of correct positional cues. The data also suggest that electrical activity of ingrowing mossy fibers down regulates nitric oxide synthase expression and plays an important role in the generation of granule cell compartments. These compartments may contribute to the functional organization of the cerebellar cortex.