Morphological and electrophysiological characteristics of dopaminergic and non-dopaminergic neurons in the substantia nigra and their postsynaptic responses to stimulation of the tegmental pedunculopontine nucleus were studied in rat organotypic triple cultures. These cultures consisted of the subthalamic nucleus explant, ventral mesencephalic explant, inclusive of the substantia nigra and the mesopontine tegmentum explant, inclusive of the tegmental pedunculopontine nucleus, prepared from one- to two-day-old rats. Intracellular sharp and whole-cell recordings were obtained from three- to eight-week-old organotypic cultures. Recorded neurons were identified as dopaminergic and non-dopaminergic neurons with tyrosine hydroxylase immunohistochemistry. Dopaminergic neurons had long duration action potentials, prominent afterhyperpolarization, time-dependent inward and outward rectification and strong frequency adaptation. Spontaneous firing patterns varied from regular, irregular to burst firing. Non-dopaminergic neurons had short duration action potentials, in general no rectifying currents, and maintained high firing frequencies. Spontaneous firing patterns in these neurons were irregular or burst firing. Morphological analysis of the recorded neurons labeled with neurobiotin revealed that non-dopaminergic neurons had more extensive arborization of higher-order dendrites than dopaminergic neurons. Dopaminergic and non-dopaminergic neurons receive glutamatergic and cholinergic excitatory inputs from the tegmental pedunculopontine nucleus. These results indicate that morphological and electrophysiological characteristics of substantia nigra neurons in the organotypic culture are generally similar to those reported in in vitro slice and in vivo studies. However, spontaneous activities of dopamine neurons observed in the organotypic culture preparation more closely resemble those in in vivo preparation compared to in vitro preparation.