The mesencephalic locomotor region is defined as a functional region sending signals to the spinal cord generators of rhythmical limb movements for locomotion. It has been shown that the mesencephalic locomotor region plays a critical role in locomotion initiated from the nucleus accumbens or from the subpallidal region. However, there are conflicting data on whether synaptic input from the nucleus accumbens--subpallidal region to the mesencephalic locomotor region mediates locomotion. The purpose of the study was to determine the role of synaptic input to different subregions of the mesencephalic locomotor region in locomotion induced by injecting dopamine into the nucleus accumbens or by injecting picrotoxin into the subpallidal region in freely behaving rats. Synaptic transmission in the mesencephalic locomotor region was eliminated by excitotoxic lesions or was reversibly interrupted by injecting cobalt chloride, which can block synaptic transmission. Excitotoxic lesions or injections of cobalt into subregions of the mesencephalic locomotor region significantly decreased, although did not completely block, locomotion. The most effective sites for cobalt- and lesion-induced reduction in locomotion were consistent with localization of the mesencephalic locomotor region. Effective sites for cobalt and lesions markedly overlapped but were not identical. The results indicate that synaptic transmission within the mesencephalic locomotor region contributes to dopamine- or picrotoxin-induced locomotion.