During development of the eye, invagination of the optic cup gives rise to a double layered neuroepithelium, part of which differentiates into the retinal pigment epithelium (RPE). The molecular mechanisms which control differentiation of the RPE are not known. The present study was undertaken to determine 1) when induction of the RPE has occurred in chicken embryos and 2) to investigate whether contact with the presumptive neural retina (NR) is required for RPE differentiation. In order to investigate when RPE induction has occurred, early expression of two genes involved in pigmentation were investigated. Digoxigenin-labeled tyrosinase and tyrosinaserelated protein-2 (TRP-2) riboprobes were synthesised and used in ISH reactions on embryonic eye tissue. Tyrosinase transcripts were first detected at stage 19.5 (70-71 hours) and TRP-2 transcripts were detected a few hours earlier at stage 18.5 (67-69 hours) of embryonic development. These results indicate that induction has occurred by stage 18.5, approximately ten hours before distinct granules are visible in the RPE. The tyrosinase and TRP-2 transcripts were always localised first in the optical axis of the eye in the region where pigment granules are first present. This indicates that differentiation of the RPE proceeds from the optical axis of the eye cup outwards towards the periphery and that induction of the RPE may also proceed in this direction. To determine whether the presumptive NR is required for RPE induction, synthetic barriers were inserted into the uninvaginated optic vesicle of chicken embryos at stage 11 (40-45 hours) of development. The embryos were cultured in vitro until the optic vesicle had invaginated and sectioned to locate the barrier. Results suggest that contact with the presumptive NR may not be necessary for RPE induction.