Neurons of the dorsal raphe nucleus exhibit intrinsic pacemaker potentials (gradual interspike depolarizing ramps) enabling them to sustain spontaneous rhythmic activity in the absence of synaptic interactions. A depolarizing prepotential (PP) has been observed in these cells, which appears to trigger the spike toward the end of the pacemaker cycle. The purposes of this study, carried out in the rat dorsal raphe nucleus brain slice preparation, were to (1) determine the ionic nature of the PP, (2) investigate its time- and voltage-dependent properties, and (3) investigate the possible modulation of the underlying conductance by the α1-agonist phenylephrine and by serotonin (5-HT), agents that modify dorsal raphe pacemaker activity. During intracellular recording under current clamp, PPs were completely and reversibly blocked by divalent cations indicating that Ca2+ carries a significant portion of the current causing the PPs. Ni2+ specifically inhibited the PP with no effect on high-threshold (−40 mV) Ca2+ spikes or the Ca2+ activated K+ conductance in these neurons. Activation threshold for the PP was found to be approximately −60 mV. Priming by hyperpolarization allowed removal of inactivation (de-inactivation) of the PP in a time- and voltage-dependent manner, with maximal PPs accompanying hyperpolarizing pulses to −90 mV and the de-inactivation beginning to occur between −65 and −75 mV. Single-electrode voltage-clamp experiments demonstrated a region of negative-slope conductance between −60 and −50 mV, which corresponds to the range of PP activation. The results of this study are consistent with a lowthreshold Ca2+ conductance underlying the PP whose role is to enable the membrane potential to rebound to action potential threshold at the end of the pacemaker cycle; neither phenylephrine nor 5-HT directly affected this inward current.