Retinoic acid receptor (RAR) signaling is required for morphogenesis of the ventral optic cup and closure of the choroid fissure, but the mechanisms by which this pathway regulates ventral eye development remain controversial and poorly understood. Although previous studies have implicated neural crest–derived periocular mesenchyme (POM) as the critical target of RA action in the eye, we show here that RAR signaling regulates choroid fissure closure in zebrafish by acting on both the ventral optic cup and the POM. We describe RAR-dependent regulation of eight genes in the neuroepithelial cells of the ventral retina and optic stalk and of six genes in the POM and show that these ventral retina/optic stalk and POM genes function independently of each other. Consequently, RAR signaling regulates ventral eye development through two independent, nonredundant mechanisms in different ocular tissues. Furthermore, the identification of two cohorts of genes implicated in ventral eye morphogenesis may help to elucidate the genetic basis of ocular coloboma in humans.