The synaptic inputs to sustained OFF-center ganglion cells of the mudpuppy retina were studied using a superfused retina-eye-cup preparation. Intra- and extracellular electrophysiological recording techniques were carried out during bath application of 2-amino-4-phosphonobutyrate (APB), a glutamate analog that selectively blocks the light responses of ON-bipolars but has minor effects on OFF-bipolar or horizontal cells. The use of APB reduced ganglion cell inputs to those arising from the OFF-bipolar channel. In this way, the existence and polarity (depolarizing vs. hyperpolarizing) of direct or indirect bipolar connections to ganglion cells was determined. Cobalt application was used to block synaptic transmission and demonstrate that APB does not have a direct excitatory action on ganglion cells. Intracellular recording experiments included the use of pulsatile and sustained current injection to evaluate the input resistance changes associated with light, the action of APB, and the excitatory, inhibitory, or disfacilitory nature of the postsynaptic potentials. Some intracellularly recorded cells were stained with horseradish peroxidase (HRP) to verify the ganglion cell origin of the recordings. The OFF-ganglion cell population of the mudpuppy appears to be a heterogeneous group of cells. Sustained OFF-ganglion cells can receive dominant inputs through either the ON- or OFF-bipolar cell pathway or through a mixture of the two. Based on the analysis of this study, we divided sustained OFF-ganglion cells into three subclasses. For one class, light causes the removal of a sustained excitatory input which originates from the OFF-bipolar channel (i.e., a light-evoked disfacilitation); a second class of cells is almost entirely driven by the ON-bipolar channel through a sustained light-evoked inhibitory input; and a third class receives both a light-evoked sustained disfacilitory input from the OFF-bipolar channel and a sustained inhibitory input through the ON-bipolar pathway. Thus the retina appears to use a variety of mechanisms that result in a common response to flashing light stimuli. The results of this study show that APB can be a powerful tool for pharmacologically deciphering the functional connections that exist between outer and inner retinal neurons.