Transmitter-like molecules are thought to influence many aspects of neuronal development, often by regulating the levels of intracellular calcium. Using the Ca2+ sensitive dye, fura-2, this study demonstrates that in the rabbit retina, ACh analogs stimulate a rise in cytosolic free Ca2+ concentration ([Ca2+]i) in many cell types, and in cells at various stages of differentiation during embryonic and neonatal development. The elevation in [Ca2+]i in cells within the ventricular zone (VZ) resulted primarily from the activation of muscarinic receptors. By contrast, the cholinergic regulation of [Ca2+]i of ganglion cells and amacrine cells, cell types which migrate to their final destinations early in fetal life, was largely mediated by nicotinic receptors. The muscarinic response of the VZ cells was mediated by the M1, rather than the M2-type of muscarinic receptor. This response was abolished in the absence of extracellular Ca2+ and in the presence of NiCl2, but it was not affected by verapamil or omega-conotoxin, thus suggesting that while Ca2+ influx occurred, it did not involve L- and N-type voltage-gated Ca2+ channels. The muscarinic response in the VZ disappeared at the end of the period of cell division in the retina, just prior to eye opening. By contrast, nicotinic-induced changes in [Ca2+]i in ganglion cells and amacrine cells persisted throughout development. Since previous studies have implicated that the precursors of ganglion cells and amacrine cells also possess muscarinic receptors (Yamashita and Fukuda, 1993), the concomitant emergence of different functional cholinergic receptor (AChR) subtypes with differentiation in vivo suggests that ACh may play diverse and temporally regulated roles in the developing retina.