Dopaminergic interplexiform amacrine cells were labeled in transgenic mice with human placental alkaline phosphatase and could therefore be identified after dissociation of the retina and used for whole-cell current and voltage clamp. In absence of synaptic inputs, dopaminergic amacrines spontaneously fired action potentials in a rhythmic pattern. This activity was remarkably robust in the face of inhibition of various voltage-dependent ion channels. It was minimally affected by external cesium or cobalt, suggesting no involvement of either the hyperpolarization-activated cation current Ih or voltage-dependent calcium channels. Inhibiting calcium-activated potassium channels by charybdotoxin or tetraethylammonium slowed the repolarizing phase of the action potentials and eliminated a slow afterhyperpolarization but had a scarce effect on the frequency of spontaneous firing. Voltage-clamp experiments showed that the interspike depolarization leading to threshold results from tetrodotoxin-sensitive sodium channels active at the interspike voltages of -60 to -40 mV. Because dopamine acts on distant targets in the retina, the pacemaker activity of dopaminergic amacrines may be necessary to ensure a tonic release of the modulator from their dendritic tree. Pacemaking is a property that this type of retinal amacrine cell shares with the dopaminergic mesencephalic neurons, but the ionic mechanisms responsible for the spontaneous firing are apparently different.