We related rod to horizontal cell synaptic transfer to glutamate release by rods. Simultaneous intracellular records were obtained from dark-adapted rod-horizontal cell pairs. Steady-state synaptic gain (defined as the ratio of horizontal cell voltage to rod voltage evoked by the same light stimulus) was 3.35 +/- 0.60 for dim flashes and 1.50 +/- 0.03 for bright flashes. Under conditions of maintained illumination, there was a measurable increment of horizontal cell hyperpolarization for each light-induced increment of rod hyperpolarization over the full range of rod voltages. In separate experiments we studied glutamate release from an intact, light-responsive photoreceptor layer, from which inner retinal layers were removed. Steady light reduced glutamate release as a monotonic function of intensity; spectral sensitivity measures indicated that we monitored glutamate release from rods. The dependence of glutamate release on rod voltage was well fit by the activation function for a high-voltage-activated, dihydropyridine-sensitive L-type calcium current, suggesting a linear dependence of glutamate release on [Ca]i in the synaptic terminal. A simple model incorporating this assumption accounts for the steady-state gain of the rod to horizontal cell synapse.