To test the hypothesis that local environmental cues regulate the expression of middle wavelength-sensitive (MWS) and short wavelength-sensitive (SWS) opsins in cone photoreceptors, we examined the development of the neonatal mouse retina in an organotypic culture system. The segregation of MWS and SWS cones into dorsal and ventral fields in the mouse retina offers an opportunity to isolate a phenotypically homogeneous population of immature cones prior to opsin expression. Retinae were harvested from mice ranging in age from birth (P0) to P18 and maintained in vitro for up to 4 weeks. Cones from newborn mice were first immunoreactive to SWS opsin-specific antibodies (OS-2 and JH455) after 5 days in vitro, which corresponds to a time course similar to that in vivo. The topographic separation of SWS cones into distinct dorsal and ventral fields was also obvious in retinal explants from newborn mice. However, the MWS opsin, identified by polyclonal antibody JH492, was expressed only in vitro when dorsal explants were harvested from P3 or older pups. Despite the absence of MWS opsin expression in newborn retinal cultures, there was no evidence of an increase in the numbers of SWS cones. To test if local diffusable cues could induce immature cones to express an aberrant opsin, dorsal and ventral retinal explants at different stages of maturation were cocultured during the incubation period. Neither the emergence of the cone fields nor the difference in the regional and temporal development of the MWS and SWS opsins was affected in these experiments. These results suggest that positional information in the retina and the opsin identity of cones is determined prior to birth and argue against the hypothesis that postnatal cones can be induced to express an aberrant opsin.