AII amacrine cells play a critical role in the high-fidelity signal transmission pathways involved with nighttime vision. The temporal properties of the light responses strongly depend on the transfer function at different synaptic stages and consequently on presynaptic calcium influx. AII light responses are complex waveforms generated by graded input, they comprise Na+-based spikes as well as a sustained component, and they are transferred to graded cone bipolar cells. It is, therefore, of interest to determine the properties of AII voltage-dependent calcium channels (VDCCs) to establish whether these cells express N-type and/or P/Q-type VDCCs, characteristic of spiking neurons, or whether they are more like graded neurons, which mostly use L-type VDCCs. We combined electrophysiological, molecular biological, and imaging techniques to characterize calcium currents and their sites of origin in mouse AII amacrine cells. Calcium currents activated at potentials more positive than -60 mV (maximally between -50 and -20 mV) and inactivated slowly. These currents were blocked by dihydropyridine (DHP) antagonists and were enhanced by the DHP agonist BayK 8644. Single-cell RT-PCR analysis of mRNA encoding for different calcium channel alpha subunits in AIIs revealed a consistent expression of the alpha1-D subunit. Calcium imaging of AII cells showed that the greatest change in intracellular calcium occurred in the lobular appendages, with minor changes being observed in the arboreal dendrites. Depolarization-induced calcium rises were also modulated by DHPs, suggesting that a particular kind of L-type VDCC, mainly localized to the lobular appendages, enables these spiking-capable neurons to release neurotransmitter in a sustained manner onto OFF-cone bipolar cells.