A dopaminergic projection from the ventral tegmental area to the ventral pallidum was identified in the rat using anterograde tract tracing and combined retrograde tracing-immunocytochemistry. The projection was found to be topographically organized such that fibers innervating the ventromedial ventral pallidum arose from neurons located along the midline nuclei of the ventral mesencephalon, including the nucleus interfascicularis and nucleus linearis caudalis. Ventral tegmental neurons situated more laterally, in the nucleus parabrachialis pigmentosus and nucleus paranigralis, projected to the ventromedial and dorsolateral ventral pallidum. The substantia nigra did not supply a major contribution to this projection. The proportion of ventral tegmental area dopaminergic neurons projecting to the ventral pallidum ranged from approximately 30% to 60%. The functional significance of the projection is indicated since intra-ventral pallidum microinjections of dopamine elicited a dose-dependent increase in locomotor activity. Furthermore, whereas pretreatment of the ventral pallidum with the GABAA agonist muscimol has been shown to attenuate opioid-induced locomotor activity elicited from the ventral pallidum, it did not attenuate the dopamine-induced motor response. Thus, while mu-opioids in the ventral pallidum may presynaptically regulate GABAergic efferents from the nucleus accumbens, it appears that the dopaminergic input directly influences the ventral pallidal output neuron which is involved in locomotion.