Topographic projections of the nervous system are essential to numerous brain functions. They arise during development as a result of encounters between projecting growth cones and particular target cells. Cellular localization of guidance cues can indicate the sequential processes involved in establishment of such topography. The map formed by retinal ganglion cells on their target nuclei has served widely as a model system to investigate mechanisms underlying the highly precise and stereotypic connectivity of the nervous system. To investigate cellular localization of guidance cues in the developing retinotectal system, a three-compartment chamber was created to delimit areas where cultured embryonic chick retinal ganglion axons and tectal cells encounter one another and guidance behavior could be readily assessed. Whereas explants from nasal retinae extended fibers across their natural target population, fibers from temporal regions of retinae failed to invade areas of growing posterior tectal cells. This preservation of relevant guidance information on living cell populations enabled an evaluation of retinal ganglion cell growth cone behavior after encounter with individual tectal cells. Posterior tectal neurons appeared selectively repulsive for temporal retinal ganglion cell growth cones, causing growth cone collapse and retraction. On the contrary, neuroepithelial cells from all regions of the tectum attenuated retinal ganglion axon extension, without inducing sudden retraction. Nasal growth cones traversed or tracked more often along neuroepithelial cells from their natural target area, potentially indicating a second set of guidance cues possibly localized to posterior glia. Together, these differential interactions suggest that development of retinotectal topography critically depends on cell-specific cues, which are distributed selectively on particular populations of target cells.