The ventral tegmental area (VTA) contains dopamine (DA) and γ-aminobutyric acid (GABA) neurons involved in motivation and behavioral state. These phenomena are also influenced by circadian factors. The goal of our studies was to examine the impulse activity of neurochemically identified VTA neurons during dark (active) vs light (rest) phases of the circadian cycle. Using extracellular single-unit recordings with juxtacellular labeling in anesthetized rats, we found multiple neuronal subpopulations including ‘novel neurons’ that selectively fired during the dark phase. These novel neurons were electrophysiologically categorized into two groups, ‘novel wide-spike’ and ‘novel thin-spike’ neurons. Characterization of novel wide-spike neurons found they were consistently non-dopaminergic and non-GABAergic [tyrosine hydroxylase (TH)(–), glutamic acid decarboxylase (GAD)(–)]. However, they were inhibited by the D2 agonist quinpirole, an effect that could be reversed by the D2 antagonist eticlopride. Physiologically, they were fast firing (mean = 18.9 ± 1.2 spikes/s), low bursting neurons (median = 6.2 ± 3.0% of spikes in bursts) with spike durations ≥ 2.0 ms, but slightly shorter than TH(+) neurons. They were also consistently non-responsive to footpad stimulation. The novel thin-spike neurons were neurochemically heterogeneous, and were located more ventrally than thin-spike neurons found during the light phase. These findings reveal previously unknown populations of VTA neurons whose activities are sensitive to diurnal phase, and whose functions may be in the temporal regulation of arousal and motivational processes.