NADPH-diaphorase-positive neurons have been demonstrated in the inner nuclear layer and ganglion cell layer of the retina of different mammalian species, but so far no experiments have been conducted to identify whether these cells are amacrine cells and/or retinal ganglion cells. We attempted to solve this problem by studying the NADPH-diaphorase-positive neurons in the hamster retina. From the NADPH-diaphorase histochemical reaction, two distinct types of neurons in the hamster retina were identified. They were named ND(g) and ND(i) cells. The ND(g) cells were cells with larger somata, ranging from 10 to 21 microns in diameter with a mean of 15.58 microns (S.D. = 2.59). They were found in the ganglion cell layer only. The ND(i) cells were smaller, with the somata ranging from 7 to 11 microns and having the mean diameter of 8.77 microns (S.D. = 1.24). Most of the ND(i) cells were found in the inner nuclear layer, and only very few could be observed in the inner plexiform layer. On average, there were 8,033 ND(g) and 5,051 ND(i) cells in the ganglion cell layer and inner nuclear layer, respectively. Two experiments were performed to clarify whether any of the NADPH-diaphorase neurons were retinal ganglion cells. Following unilateral optic nerve section, which leads to the retrograde degeneration of retinal ganglion cells, the numbers of both ND(g) and ND(i) cells did not change significantly for up to 4 months. In addition, when retinal ganglion cells were prelabeled retrogradely (horseradish peroxidase or fluorescent microspheres) and retinas were then stained for NADPH diaphorase, no double-labeled neurons were detected. These results indicated that the NADPH-diaphorase neurons in the hamster retina were the amacrine cells in the inner nuclear layer and displaced amacrine cells in the ganglion cell layer. Dendrites of the ND(g) and ND(i) cells were found to stratify in sublaminae 1, 3, and 5 of the inner plexiform layer, with a prominent staining in the sublamina 5. The possible importance of this arrangement in the rod pathway is also discussed.