We used two different methodologies to examine at what stage development retinal positional specificity is established and which molecules are responsible. The first goal was achieved by removing parts of the presumptive temporal primary optic vesicle at stage 11 (40 to 45 hr of incubation) and fate mapping of tissue with presumptive nasal properties that shifted into the wound during the events of wound-healing. Participation of the shifted tissue in the healing resulted in assembly of a temporal retina with mosaic-like projection properties, as examined by retrograde double staining of the retinal ganglion cells from the optic tectum. In addition to cells with normal temporal-rostral projections, clusters of ganglion cells with nasal-like projection identities appeared labelled within the temporal hemiretina. The number of clusters increased with the amount of resected tissue, and by almost complete ablation of the presumptive temporal anlage, a temporal hemiretina with predominantly nasal retinotectal specificity was created. These neuroanatomical results suggested that neuroepithelial cells had fixed nasal and temporal positional specificities at stage 11. To examine differences in the cells derived of either half of the eye cup, we performed biochemical one- and two-dimensional gel electrophoresis of the hemianlagen at stage 11. In addition, incorporation of 35S-methionin into newly synthesized peptides was investigated. Both techniques revealed the exclusive expression of one major and three less-abundant proteins within the presumptive nasal anlage. The most abundant of these proteins has a molecular weight of about 40 kDa and is clearly distinguishable both in gel electrophoresis and autoradiography. The asymmetric protein patterns had disappeared when the retina was analysed with the same methods at the more advanced embryonic days E4 and E6. The asymmetry in the expression of proteins in the retinal primordium may be the biochemical correlate of an early positional specification of the retinal neuroepithelium. The difference in the protein expression may explain that mixing the positionally specified cells of either origins results in projection mosaics.