even-skipped-related (evx) genes encode homeodomain-containing transcription factors that are involved in a series of developmental processes such as posterior body patterning and neurodifferentiation. Although evx1 and evx2 were not reported to be expressed during mammalian tooth development, we present here evidence that eve1, the closest paralog of evx1 in the actinopterygian lineage, is expressed during pharyngeal tooth formation in the zebrafish, Danio rerio. We have performed whole-mount in situ hybridization on zebrafish embryos and larvae ranging from 24 to 192 hours postfertilization (hpf). A detailed analysis of serial sections through the pharyngeal region of whole-mount hybridized and control specimens indicates that only dental epithelial cells express eve1. eve1 transcription was activated at 48 hpf, in the placode of the first tooth (i.e., the initiation site of tooth 4V1), and maintained in the dental epithelium throughout morphogenesis. Then, by 72 hpf, eve1 expression was restricted to the differentiating ameloblasts of the enamel organ during early differentiation stage, and this expression decreased as soon as matrix was deposited. In subsequent primary teeth (3 V1 and 5 V1) as well as in their successors (replacement teeth 4V2, 3V2, and 5V2), eve1 expression was restricted to the differentiating ameloblasts and, again, disappeared when matrix was deposited. Therefore, in the zebrafish, eve1 expression in the pharyngeal region is correlated with two key steps of tooth development: initiation and morphogenesis of the first tooth, and ameloblast differentiation of all developing teeth. Developmental Dynamics 230:727–733, 2004. © 2004 Wiley-Liss, Inc.