The creation of specific neuronal cell types within the developing brain is a critical and unsolved biological problem. Precedent from invertebrate development, and from vertebrate myogenesis and lymphogenesis, has established that cell specification often involves transcription factors that are expressed throughout the differentiation of a given cell type. In this study, we have identified in Zn2+ finger transcription factor RU49 as a definitive marker for the cerebellar granule neuron lineage. Thus, RU49 is expressed in the earliest granule cell progenitors at the rhombic lip as they separate from the ventricular zone of the neural tube to generate a secondary proliferative matrix, and it continues to be expressed in differentiating and mature granule neurons. Proliferating granule cell progenitors isolated from the rhombic lip at E14 or from the external germinal layer at P6 continue to express RU49 in vitro. Both the olfactory bulb and dentate gyrus granule cell lineages also express this factor as they are generated with the developing brain. RU49 binds a novel bipartite DNA-binding element in a manner consistent with chemical rules governing the DNA-binding specificity of this class of transcription factor. The novel biochemical properties of RU49 and its restricted expression within the three lineages of CNS granule neurons suggest that RU49 may play a critical role in their specification. Furthermore, these results raise the interesting possibility that the generation of these three neuronal populations to form displaced germinative zones within the developing brain may reflect their use of a common developmental mechanism involving RU49.