Members of the PAX family of transcription factors are candidates for controlling cell identity in the spinal cord. We have morphologically analyzed cells that express one of these transcription factors, PAX2, demonstrating multiple interneuron cell types express PAX2. Two ventral populations of PAX2-expressing interneurons in the spinal cord are marked by coexpression of the transcription factors, EN1 and EVX1. Interestingly, the expression domains of PAX2, EN1 and EVX1 in postmitotic neurons correlate closely with those of Pax6 and Pax7 in the ventricular zone, implicating these patterning genes in the regulation of PAX2, EN1 and EVX1. We show that one of these patterning genes, Pax6, is required for the correct specification of ventral PAX2+ interneurons that coexpress EN1. These results demonstrate that the early activity of patterning genes in the ventricular zone determines interneuron identity in the spinal cord.