Two types of tegmental pedunculopontine nucleus neurons have been reported previously based on their electrophysiological characteristics: type I neurons were characterized by low-threshold Ca spikes and type II neurons displayed a transient outward current. This report describes the membrane properties, synaptic inputs, morphologies and axonal projections of two subgroups of type II neurons examined in an in vitro slice preparation. Type II neurons were divided into two groups based on their spike durations: short-duration neurons with an action potential duration of 0.7-1.5 ms and long-duration neurons with an action potential duration of 1.6- 2.9 ms. Choline acetyltransferase immunohistochemistry combined with biocytin labeling indicated that 56% of short-duration neurons and 61% of long-duration neurons were immunopositive for choline acetyltransferase. Short-duration neurons had a high input resistance and the capacity to discharge with high frequency. By contrast, long-duration neurons had a low input resistance and low firing frequency and upon current injection displayed an accommodation (spike-frequency adaptation) before reaching a steady firing frequency. Microstimulation of the substantia nigra pars compacta evoked antidromic responses in both short-duration neurons (n=5/14, 36%) and long-duration neurons (n=20/39. 51%). Stimulations of the subthalamic nucleus and the substantia nigra pars reticulata induced in these neurons excitatory and inhibitory postsynaptic potentials, respectively. Short-duration neurons were dispersed equally throughout the extent of the tegmental pedunculopontine nucleus area, while long-duration neurons were located more in the rostral tegmental pedunculopontine nucleus. Short-duration neurons were small with two to four thin primary dendrites. Long-duration neurons were medium to large with three to six thick primary dendrites. Cell size was positively correlated with spike duration and axonal conduction velocity, but negatively with input resistance and spontaneous firing frequency. Both groups of neurons had ascending (toward thalamus, pretectal areas and tectum) and descending (toward pontomedullary reticular formation) axons in addition to nigropetal axons. Ascending axons were observed in 75% (6/8) of short-duration neurons and in 45% (15/33) of long-duration neurons, while nigropetal axons were observed in 50% (4/8) of short-duration neurons and in 76% (25/33) of long-duration neurons. These results suggest that the tegmental pedunculopontine nucleus cholinergic projection system is composed of heterogeneous populations of neurons in terms of electrophysiological and morphological characteristics as well as their distribution patterns in the nucleus.