A culture model has been established to study the gradual development of tight junctions during the embryogenesis of the chick retinal pigment epithelium. This study asks how closely the culture model reflects normal development and how the composition, structure and function of embryonic tight junctions are affected by the apical and basal environments. The study focused on the expression of claudins, the fine-structure of tight junctional strands and the transepithelial electrical resistance. Between embryonic days 7 and 14, patches of junctional strands gradually expanded and coalesced to form a continuous junction, in vivo. Although there was a corresponding increase in claudin expression, different claudins appeared at different times. In culture, the apical and basal environments acted synergistically to promote a continuous network of tight junctions with higher electrical resistance. Independently, pituitary extract or the secretory products of either embryonic fibroblasts or the retina promoted the formation of tight junctions. In combination, three effects were identified. With basally placed fibroblast conditioned medium, apical retinal medium increased transepithelial electrical resistance by affecting structure alone. With basally placed pituitary extract, apical retinal conditioned medium increased transepithelial electrical resistance by affecting structure and by modulating claudin expression in a manner that was consistent with development in vivo. Although embryonic day 7 and 14 cultures in retinal medium exhibited similar structure, the transepithelial electrical resistance of the embryonic day 14 cultures was higher. This higher transepithelial electrical resistance correlated with differences in claudin expression and localization. Therefore, this experimental model can isolate the effects of retinal secretions on structure and claudin expression, and can help us to determine how claudins affect function when structure is held constant.