We examined the spatial expression of the relaxin-3 gene in the developing zebrafish brain, one of the vertebrate model systems in which this gene has been identified. Until the pharyngula stage, the gene is expressed diffusely in the brain, where, starting at about 40 hpf, the transcripts appear restricted in a midbrain cell cluster of the periaqueductal gray. Later, at 72 hpf, the transcripts are still evident in that cluster and distributed in a larger cell number; at this stage, the gene is also expressed posteriorly, in a smaller cell group that, as we report for the first time, could be homologous to mammalian nucleus incertus. The gene expression persists in both cell clusters at 96 hpf. This pattern indicates both conserved and divergent expression features of the relaxin-3 gene among developing zebrafish and rat brains, where only scattered cells express the gene in the periaqueductal gray. Developmental Dynamics 237:3864–3869, 2008. © 2008 Wiley-Liss, Inc.