Neurons originating in the ventral tegmental area are thought to play a key role in the formation of addictive behaviors, particularly in response to drugs such as cocaine and opioids. In this study we identified different populations of ventral tegmental area neurons by the pharmacology of their evoked synaptic potentials and their response to dopamine, 5-hydroxytryptamine and opioids. Intracellular recordings were made from ventral tegmental area neurons in horizontal slices of guinea-pig brain and electrical stimulation was used to evoke synaptic potentials. The majority of cells (61.3%) hyperpolarized in response to dopamine, depolarized to 5-hydroxytryptamine, failed to respond to [Met]5enkephalin and exhibited a slow GABAB-mediated inhibitory postsynaptic potential. A smaller proportion of cells (11.3%) hyperpolarized in response to [Met]5enkephalin, depolarized to 5-hydroxytryptamine, failed to respond to dopamine and did not exhibit a slow inhibitory postsynaptic potential. These two groups of cells corresponded to previously described "principal" and "secondary" cells, respectively. A further group of cells (27.4%) was identified that like the principal cells, hyperpolarized to dopamine. However, these "tertiary cells" also hyperpolarized to both 5-hydroxytryptamine and [Met]5enkephalin and exhibited a slow, cocaine-sensitive 5-hydroxytryptamine(1A)-mediated inhibitory postsynaptic potential. When principal and tertiary cells were investigated immunohistochemically, 82% of the principal cells were positive for tyrosine hydroxylase compared with only 29% of the tertiary cells. The 5-hydroxytryptamine innervation of both these cell types was investigated and a similar density of putative contacts was observed near the somata and dendrites in both groups. This latter finding suggests that the existence of a 5-hydroxytryptamine-mediated inhibitory postsynaptic potential in the tertiary cells may be determined by the selective expression of 5-hydroxytryptamine receptors, rather than the distribution or density of the 5-hydroxytryptamine innervation. We conclude that tertiary cells are a distinct subset of ventral tegmental area neurons where cocaine and mu-opioids both mediate inhibition.