A novel POU-domain protein, retina-derived POU-domain factor-1 (RPF-1), has been identified through the isolation of cDNA and genomic DNA clones. In the adult, RPF-1 is expressed only within the CNS, where its expression is restricted to the medical habenulla, to a dispersed population of neurons in the dorsal hypothalamus, and to subsets of ganglion and amacrine cells in the retina. The human RPF-1 gene spans > 125 kb and gives rise to multiple differentially spliced transcripts. In the human retina, the most abundant mRNA isoforms are derived from an alternate splicing event that inserts an evolutionarily conserved peptide of 36 amino acids into the DNA recognition helix of the POU- specific domain. In vitro, the RPF-1 POU domain lacking the insert binds to a consensus Oct-1 binding site, whereas the alternately spliced POU domain does not. RPF-1 protein first appears in the developing mouse retina at e11, where it localizes to neuroblasts that have recently migrated from the mitotic zone to the future ganglion cell layer. These data suggest that RPF-1 is likely to be involved in early steps in the differentiation of amacrine and ganglion cells.