Purpose. A majority of retinal amacrine cells (Acs) either contain the inhibitory amino acid neurotransmitter GABA or glycine (GLY). In the mammalian retina, a subpopulation of glycinergic amacrine cells contains the calcium binding protein, parvalbumin (PARV). Double-label immunohistochemistry was used to determine if GLY and PARV were co-localized in the goldfish retina. Methods. Eyecups were fixed in 4% paraformaidehyde and 0.5% acrolein for 1 hr, treated with NaBH for 45 min, and cryoprotected in 30% sucrose overnight. Cryostat sections were cut at 10μm and mounted on APTEX-treated slides. In single labelling studies, sections were incubated in mouse∞PARV (Σ ; 1:100) or rabbit∞GLY (Chemicon; 1:100) overnight and visualized using goat∞mouse- or goat∞rabbit-Texas Red (BioCan; 1:100). Flatmounts were prepared by isolating retinas from dark-adapted fish, fixing as described above, incubating the free-floating retina in antisera overnight, washing, incubating in secondary fluorescent antisera for 8 hrs, and examined by confocal microscopy. Double-labelling experiments were performed by mixing primary antisera (final dilutions: 1:100), incubating sections overnight, washing, and incubating in a mixture of goat∞rabbit-FITC and goat∞mouse-Texas Red. Results. Two populations of PARV-IR amacrine cells were identified, having different staining intensities. The brightly-labeled cells were 8.8±0.6μm in diameter and were located in the first tier of cells in the INL. A single, thin primary process projected to the 60% depth of the IPL where it ramified into a narrowly stratified, beaded plexus. Confocal micrscopy revealed that the cells made close contacts with MBb1 bipolar cell axon terminals. These PARV cells were uniformly distributed over the surface of the retina, forming a regular mosaic (density: 51/mm2; Nearest Neighbor Distance: 111±16μm). Double-labeling studies revealed that 100% of these cells also contained GLY and were not GABA-1R. The dimly-labeled PARV cells were co-localized with both GLY (59%) and GABA (19%) or not labeled by either antisera (22%), suggesting that the dimly-labeled PARV cells are a heterogenous population. Conclusions. These studies demonstrate that glycinergic amacrine cells in the fish retina do not consist of a homogenous population of diffuse amacrine cells. The subpopulation of PARV/GLY cells form a regular array of amacrine cells narrowly stratified at the middle of the IPL.