MATH1 is a neural-specific basic helix-loop-helix transcription factor. Members of this family of transcription factors are involved in the development of specific subsets of neurons in the developing vertebrate nervous system. Here we examine the cells expressing MATH1 with respect to their proliferative state and co-expression of cell-type-specific differentiation markers. We localize the MATH1 protein to the nucleus of cells in the dorsal neural tube and the external germinal layer (EGL) of the developing cerebellum. Using double-label immunofluorescence, we demonstrate that MATH1-expressing cells span both the proliferating and the differentiating zones within the dorsal neural tube, but within the EGL of the cerebellum are restricted to the proliferating zone. The early differentiating MATH1-expressing cells in the dorsal neural tube co-express TAG-1, DCC-1 and LH2, markers of dorsal commissural interneurons. In addition, transgenic mice with lacZ under the transcriptional control of MATH1-flanking DNA sequences express beta-galactosidase specifically in the developing nervous system, in a manner that mimics subsets of the MATH1-expression pattern, including the dorsal spinal neural tube. Expression of the MATH1/lacZ transgene persists in differentiated dorsal commissural interneurons. Taken together, we demonstrate MATH1 expression in a differentiating population of neuronal precursors in the dorsal neural tube that appear to give rise specifically to dorsal commissural interneurons.