AbstractThe indoleamine‐accumulating amacrine cells of the rabbit retina are wide‐field and numerous. They form a dense plexus in sublamina 5 of the inner plexiform layer where they make reciprocal synapses with rod bipolar cells. To provide a quantitative test for the colocalization of serotonin (5‐HT) and γ‐aminobutyric acid (GABA) in the rabbit retina, we designed two parallel double‐label experiments. In the first series, the indoleamine‐accumulating cells were labeled with 5,7‐dihydroxytryptamine (5,7‐DHT), which was subsequently visualized by photooxidation in the presence of diaminobenzidine. This was combined with autoradiography for 3H‐muscimol. In the second and complementary series, 3H‐5‐HT uptake was combined with postembedding GABA immunocytochemistry. These two experiments provided essentially identical results: over 98% of the indoleamine‐accumulating amacrine cells were double‐labeled. This means that, within the limit of experimental error, all the indoleamine‐accumulating amacrine cells are GABAergic. The indoleamine‐accumulating amacrine cells account for 15–20% of a large diverse group of GABA amacrine cells. In addition, the rare type 3 indoleamine‐accumulating cells and fine processes running in the optic fiber layer were double‐labeled. If there is insufficient 5‐HT to support a transmitter role in the rabbit retina, our results suggest that the indoleamine‐accumulating cells may use GABA as a neurotransmitter. Thus, rod bipolar cells, in common with other bipolar cell types, receive extensive negative feedback at GABA‐mediated reciprocal synapses. © 1992 Wiley‐Liss, Inc.