The developing eye is known to be rich in retinoic acid (RA), and perturbations in RA levels during formation of the optic primordia, as well as RA receptor mutations, cause retinal malformations, especially in ventral eye regions. To test the hypothesis that RA plays a role in the establishment of ventral retinal characteristics, we examined several dorsal and ventral ocular markers in RA-treated zebrafish. The optic stalk represents the ventral-most region of the early eye field. During normal development, the optic stalks constrict, decreasing in width and are gradually replaced by the optic nerve. Systemic high RA levels cause an expansion in the optic stalk with an increased cell content and a patent lumen. In addition, the stalks do not constrict and persist into later stages of development indicating an enhancement of early ventral eye characteristics. Expression of the transcription factor pax[b], normally confined to the ventral retina, expands into the dorsal retina following RA treatment, whereas msh[c], normally expressed in the dorsal retinal pole, disappears. Activity of an aldehyde dehydrogenase that normally occupies the dorsal third of the retina is reduced or abolished following high systemic RA. When a localized RA source, an RA-soaked bead, is placed next to the developing eye, a fissure resembling the choroid fissure appears in the eye facing the bead. Taken together, these observations suggest that RA is involved in the determination of the ventral retina.