The ventral tegmental area (VTA) and in particular VTA dopamine (DA) neurons are postulated to play a central role in reward, motivation and drug addiction. However, most evidence implicating VTA DA neurons in these functions is based on indirect electrophysiological characterization, rather than cytochemical identification. These physiological criteria were first established in the substantia nigra pars compacta (SNc), but their validity in the VTA is uncertain. In the current study we found that while 88 +/- 2% of SNc neurons labelled by the neuronal marker NeuN were co-labelled for the catecholamine enzyme tyrosine hydroxylase (TH), a much smaller percentage (55 +/- 2%) of VTA neurons co-expressed TH. In addition, using in vitro whole-cell recordings we found that widely accepted physiological criteria for VTA DA neurons, including the hyperpolarization-activated inwardly rectifying non-specific cation current (I(h)), spike duration, and inhibition by DA D2 receptor agonists, do not reliably predict the DA content of VTA neurons. We could not distinguish DA neurons from other VTA neurons by size, shape, input resistance, I(h) size, or spontaneous firing rate. Although the absence of an I(h) reliably predicted that a VTA neuron was non-dopaminergic, and I(h)(-) neurons differ from I(h)(+) neurons in firing rate, interspike interval (ISI) standard deviation, and ISI skew, no physiological property examined here is both sensitive and selective for DA neurons in the VTA. We conclude that reliable physiological criteria for VTA DA neuron identification have yet to be determined, and that the criteria currently being used are unreliable.