Amacrine cells in the mammalian retina are famously diverse in shape and function. Here, we show that two wide-field GABA amacrine cells, S1 and S2, have stereotyped synaptic contacts with the appropriate morphology and distribution to perform specific functions. S1 and S2 both supply negative feedback to rod bipolar terminals and thus provide a substrate for lateral inhibition in the rod pathway. Synapses are specialized structures, and the presynaptic compartment is normally characterized by a swelling or varicosity. Each S1 amacrine cell has approximately 280 varicosities, whereas an S2 cell has even more, approximately 500 per cell. Confocal analysis shows that essentially all varicosities aggregate around rod bipolar terminals where they are apposed by postsynaptic GABA receptors. Each rod bipolar terminal is contacted by varicosities from approximately 25 different S1 and 50 different S2 amacrine cells. In fact, rod bipolar cells are the only synaptic target for S1 and S2 amacrine cells: all of the output from these two wide-field GABA amacrine cells goes to rod bipolar terminals. It has long been a puzzle why two amacrine cells, apparently with the same connections, are required. However, an analysis of the distribution of varicosities suggests that S1 and S2 amacrine cells provide different signals. S2 amacrine cells dominate within 200 mu from a rod bipolar terminal and can provide an inhibitory input with spatial characteristics that match the size of the surround signal recorded from AII amacrine cells in the rod pathway. In contrast, the larger, better-coupled S1 amacrine cells may provide a more distant network signal.