Retinal ganglion cells (RGCs) form the topographic connection between retina and optic tectum in the developing avian embryo. In vitro, neurons with the morphological traits and marker expression of RGCs were found both in single-cell cultures from embryonic day (E) 6 chick retina and in retinal cell lines derived from E3.5 quail retina. Rapid and substantial differentiation of RGC-like cells could be induced in the lines by addition of fibroblast growth factor aFGF or bFGF. RGC-like cells were examined with respect to their target discrimination properties as single cells in the stripe carpet assay. In this assay system, alternating stripes of membrane vesicles prepared from the anterior and posterior tectum are offered to growing axonal processes as a substrate. Temporal RGC-like cells, both primary cells prepared from the temporal retina and immortalized cells of those retinal lines derived from the temporal retina, avoid stripes of membrane vesicles from posterior tectum; they prefer to grow on membrane vesicles from the anterior tectum, which is their in vivo target. Nasal RGC-like cells did not exhibit a target preference, in accordance with previous findings. Together the experiments show that target preference of RGCs is a cell-autonomous and heritable mechanism that is determined early and is position-dependent.