We are investigating the differentiation of the major subtypes of horizontal cell in the rabbit retina in order to learn more about developmental controls responsible for the variety of neuronal phenotypes. Immunohistochemistry with anti-neurofilament and anti-calbindin-D antibodies, followed by epoxy resin embedding, has facilitated study of these neurons. In the mature rabbit retina, axonless (A-type) horizontal cells reacted strongly in procedures using either antibody; short axon (B-type) somas did not show a reaction with anti-neurofilament antibodies and stained moderately using anti-calbindin antibodies. In the immature neonatal retina the somas of all the horizontal cells seemed to be similar with regard to general morphology, but two populations could be distinguished on the basis of immunostaining. Some, identified as A-type horizontal cells (by comparison with mature retina), were stained using either antibody. Interspersed among these were similar cells with no detectable immunoreactivity, identified as B-type horizontal cells. By the end of the first postnatal week, faint reactivity to anti-calbindin-D was present in the somas of B-type horizontal cells; they stained moderately throughout the rest of the period studied. Thus differences in immunostaining indicate that the two horizontal cell subpopulations are established early in the rabbit, though some other distinguishing characteristics emerge only gradually as the retina matures. These results suggest that in mammals the determination of phenotypic subtype occurs early, possibly at the time that the cell is specified as a horizontal neuron, or shortly thereafter.