The basic helix-loop-helix transcription factor Neurogenin2 (NGN2) is expressed in distinct populations of neural progenitor cells within the developing central and peripheral nervous systems. Transgenic mice containing ngn2/lacZ reporter constructs were used to study the regulation of ngn2 in the developing spinal cord. ngn2/lacZ transgenic embryos containing sequence found 5' or 3' to the ngn2 coding region express lacZ in domains that reflect the spatial and temporal expression profile of endogenous ngn2. A 4.4-kb fragment 5' of ngn2 was sufficient to drive lacZ expression in the ventral neural tube, whereas a 1.0-kb fragment located 3' of ngn2 directed expression to both dorsal and ventral domains. Persistent -gal activity revealed that the NGN2 progenitor cells in the dorsal domain give rise to a subset of interneurons that send their axons to the floor plate, and the NGN2 progenitors in the ventral domain give rise to a subset of motor neurons. We identified a discrete element that is required for the activity of the ngn2 enhancer specifically in the ventral neural tube. Thus, separable regulatory elements that direct ngn2 expression to distinct neural progenitor populations have been defined.