Specification of spinal cord neurons is regulated by several different transcription factors. In this study, we analyze expression and regulation of the transcription factor iro3 in zebrafish spinal cord. In addition to its broad expression in the progenitor domain of intermediate spinal cord, iro3 is also expressed in postmitotic ventral neurons, starting at early somitogenesis stages. Initially, this expression is only in two primary motoneurons, CaP and VaP, but by 24 hr postfertilization, iro3 is expressed by all classes of zebrafish spinal motoneurons as well as by a ventral interneuron called VeLD. iro3 expression in the progenitor domain of intermediate spinal cord is regulated independently from its expression in ventral neurons. Hedgehog (Hh) signaling is unnecessary for iro3 expression in intermediate spinal cord, but it is required to repress iro3 expression in the progenitor domain of ventral spinal cord. We also show that the basic helix-loop-helix transcription factor Olig2 is required for repression of iro3 expression in the progenitor domain of ventral spinal cord. We discuss our findings in the context of previous studies, suggesting that iro3 represses formation of motoneurons and promotes formation of interneurons. Developmental Dynamics 232:140–148, 2005. © 2004 Wiley-Liss, Inc.