The central connections of retinal ganglion cells are retinotopically organized, producing a "map" of the retina on the surface of the optic tectum. Exactly how and when individual ganglion cells develop the position-dependent properties (termed locus specificities) subserving formation of the map is unknown, but the positional information that each ganglion cell will use in this process is specified in the early Xenopus embryo during a critical period at stages 28-32. We report two methods for isolating eye primordia from the axial cues of the animals during this critical period and for then allowing the eyes to form retinotectal connections in a carrier embryo. The results show that, as early as optic vesicle stages 22-23, the eyes already contain orthogonal reference axes, that positional information can be specified with respect to these axes in vitro, and that the specification process itself may only entail a transition from a reversible to an irreversible state.