We have investigated the morphology of the NOS-like immunoreactive neurons and their synaptic connectivity in the rat retina by immunocytochemistry using antisera against nitric oxide synthase (NOS). In the present study, several types of amacrine cells were labeled with anti-NOS antisera. Type 1 cells had large somata located in the inner nuclear layer (INL) with long and sparsely branched processes ramifying mainly in stratum 3 of the inner plexiform layer (IPL). Somata of type 2 cells with smaller diameters were also located in the INL. Their fine processes branched mostly in stratum 3 of the IPL. A third population showing NOS-like immunoreactivity was a class of displaced amacrine cells in the ganglion cell layer (GCL). Their soma size was similar to that of the type 1 cells; however, their processes stratified mainly in strata 4 and 5 of the IPL. Labeled neurons were evenly distributed throughout the retina, and the mean densities were 57.0 ± 9.7 cells/mm2 for the type 1 cells, 239.3 ± 43.4 cells/mm2 for the type 2 cells and 121.2 ± 27.5 cells/mm2 cells for the displaced amacrine cells. The synaptic connectivity of NOS-like immunoreactive amacrine cells was identified in the IPL by electron microscopy. NOS-labeled amacrine cell processes received synaptic input from other amacrine cell processes and bipolar cell axon terminals in all strata of the IPL. The most frequent postsynaptic targets of NOS-immunoreactive amacrine cells were other amacrine cell processes. Ganglion cell dendrites were also postsynaptic to NOS-like immunoreactive neurons in both sublaminae of the IPL. Synaptic outputs onto bipolar cells were observed in sublamina b of the IPL. In addition, a few synaptic contacts between labeled cell processes were observed. Our results suggest that NOS immunoreactive cells may be modulated by other amacrine cells and ON cone bipolar cells, and act preferentially on other amacrine cells.