This study describes the identification and differentiation of neonatal rat retinal cells in monolayer cultures. A panel of monoclonal antibodies was used as a molecular probe of both cell type and developmental stage. Previously described cell-type specific monoclonal antibodies were used to label rod photoreceptors, horizontal cells, amacrine cells or ganglion cells. Two new antibodies that react with rat retina are described. The first, RET-G7, reacts with a cytoplasmic antigen of Muller glia, astrocytes and some horizontal cells. The second, RET-B2, reacts with bipolar cells and photoreceptor inner segments. Two main findings are presented. The first is that each of the major subclasses of retinal neurons have been unambiguously identified in these cultures. The morphology of some subclasses was very characteristic. All photoreceptors, as defined by reactivity with antibody RET-P1, were small spherical cells with one or fewer processes. Horizontal cells, as defined by reactivity with antibody B-1, were large with a characteristic multipolar network of processes. Bipolar and amacrine cells, on the other hand, were of similar size and could only be distinguished on the basis of immunocytochemical labeling. The second finding is that while RET-B2 antigen appeared on bipolar and photoreceptor cells after about 5 days in culture, several Muller cell and photoreceptor antigens were not expressed in monolayer cultures. The results suggest that the expression of some molecules in culture is the result of properties intrinsic to the cells whereas expression of others depends upon extrinsic factors or cell interactions that may not be present in monolayer cultures.