Horizontal cells modulate the output of the photoreceptor to bipolar cell synapse, thereby providing the first level of lateral information processing in the vertebrate retina. Because horizontal cells do not generate sodium-based action potentials, calcium is likely to play an important role for graded potential changes as well as for intracellular events involved in the modulatory role of horizontal cells within the retinal network. Therefore we wanted to determine how the activation of glutamate receptors, voltage-gated calcium channels, and release of calcium from internal stores shape the calcium signal in horizontal cells. All horizontal cells responded to depolarizing voltage steps with sustained inward currents, which activated at around -20 mV, reached a peak amplitude of -79.1 pA at 5 mV, and reversed sign at around 66 mV. The current was insensitive to tetrodotoxin, and it was partially blocked by the L-type channel antagonists verapamil and nifedipine. The N-type channel blocker omega-conotoxin GVIA induced an additional reduction of current amplitudes. Calcium influx through ionotropic glutamate receptors was mediated by both AMPA and kainate but not by N-methyl-D-aspartate receptors. Two agonists at group I metabotropic glutamate receptor, trans-1-amino-1,3-cyclopentanedicarboxylic acid and quisqualate, had no effect. However, intracellular calcium was increased by caffeine, indicating release of calcium from internal stores via ryanodine receptors. These data show that intracellular calcium in horizontal cells is regulated by voltage-dependent L- and N-type calcium channels, ionotropic AMPA and kainate receptors, and release of calcium from internal stores after activation of ryanodine receptors.