A cone photoreceptor releases glutamate at ribbons located atop narrow membrane invaginations that empty onto a terminal base. The unique shape of the cone terminal suggests that there are two transmitter microenvironments: within invaginations, where concentrations are high and exposures are brief; and at the base, where concentrations are low and exposure is smoothed by diffusion. Using multicell voltage-clamp recording, we show that different subtypes of Off bipolar cells sample transmitter in two microenvironments. The dendrites of an AMPA receptor-containing cell insert into invaginations and sense rapid fluctuations in glutamate concentration that can lead to transient responses. The dendrites of kainate receptor-containing cells make basal contacts and respond to a smoothed flow of glutamate that produces sustained responses. Signaling at the cone to Off bipolar cell synapse illustrates how transmitter spillover and synapse architecture can combine to produce distinct signals in postsynaptic neurons.