Retinal cells that respond selectively to light onset or offset are segregated into On and Off pathways. Here, we describe the development of cone bipolar cells whose axonal arbors at maturity synapse onto ganglion cell dendrites confined to On and Off strata of the inner plexiform layer (IPL). In particular, we sought to determine whether the formation of this segregated pattern is dependent on the presence of ganglion cells. Developing bipolar cells were visualized using an antibody against recoverin, the calcium binding protein that labels On and Off cone bipolar cells in the adult rat retina. Recoverin-positive cells were apparent in the ventricular zone on the day of birth [postnatal day 0 (P0)], before bipolar cells begin to migrate to the inner nuclear layer. Two distinct strata were first apparent in the IPL at P8, with the Off pathway maturing earlier than the On pathway. There was no indication of exuberant bipolar cell projections. Throughout development, there were also a small number of recoverin-positive cells of unknown origin in the ganglion cell layer. To assess whether the formation of On and Off cone bipolar cell projections is dependent on the presence of ganglion cells, these target neurons were eliminated by unilateral section of the optic nerve. This was done on the day of birth, resulting in a total loss of ganglion cells 5-6 d before bipolar cell axons innervate the IPL. In retinas with optic nerve sections, On and Off cone bipolar cells were present, albeit at a lower than normal density, and the axonal arbors of these interneurons were organized into two distinct strata. This indicates that ganglion cells are not essential for the formation of segregated On and Off bipolar cell inputs. These results lend support to the hypothesis that specific ingrowth patterns of bipolar cell terminal arbors could regulate the formation of stratified retinal ganglion cell dendrites.