Single-unit recording combined with iontophoresis of dopamine, GABA, and glutamate was used in awake, unrestrained rats to characterize the electrophysiological and receptor properties of neurons in the ventral tegmental area under naturally occurring behavioural conditions. All isolated ventral tegmental area units (n=90) were analysed and compared with cells (n=58) recorded from dorsally adjacent areas of the pre-rubral area and red nucleus. Two distinct neuronal groups were identified in the ventral tegmental area: units with triphasic, long-duration spikes (78/90) and units with biphasic, short-duration spikes (12/90). Although all long-spike units discharged in an irregular, bursting pattern with varying degrees of within-burst decrements in spike amplitude, they could be further subdivided into at least three distinct subgroups. Type I long-spike units (36/78) discharged at a relatively slow and stable rate (mean: 6.03 imp/s; range: 0.42-15.78) with no evident fluctuations during movement. These cells were inhibited by dopamine and GABA and responded to glutamate with a low-magnitude excitation accompanied by a pronounced decrement in spike amplitude and a powerful rebound inhibition. Type II long-spike units (23/78) had relatively high and unstable discharge rates (mean: 22.82 imp/s; range: 4.42-59.67) and showed movement-related phasic activations frequently followed by partial or complete cessation of firing. Some Type II cells (4/9) were inhibited by dopamine, but all were excited by glutamate at very low currents (0-10 nA). With an increase in current, the glutamate-induced excitation often (18/22) progressed into a cessation of firing. All these cells were inhibited by GABA followed by a strong rebound excitation (8/9), which also frequently (6/8) resulted in cessation of firing. Type III long-spike units (19/78) had properties that differed from either Type I or Type II cells, including a lack of spontaneous firing (5/19). Short-spike ventral tegmental area units were either silent (4/12) and unresponsive to dopamine and GABA or spontaneously active (range: 0.89-34.13 imp/s) and inhibited by GABA and, in some cases (2/8). by dopamine; all were phasically activated during movement and glutamate iontophoresis. It appears that ventral tegmental area neurons, including those with long-duration spikes, do not comprise a uniform population in awake, unrestrained rats. Type I, long-spike units match the characteristics of histochemically-identified dopamine neurons, and they appear to express dopamine autoreceptors, which may explain the relatively slow, stable rate of activity and the limited responsiveness to excitatory inputs. Although the nature of the other long-spike units in our sample is unclear, they may include dopamine neurons without autoreceptors as well as non-dopamine cells. The heterogeneity of ventral tegmental area neurons is an important consideration for further attempts to assess the role of the mesocorticolimbic dopamine system in motivated behaviour.