The mesencephalic locomotor region (MLR) plays a crucial role in locomotor control. In vertebrates, stimulation of the MLR at increasing intensities elicits locomotion of growing speed. This effect has been presumed to result from higher brain inputs activating the MLR like a dimmer switch. Here, we show in lampreys (Petromyzon marinus) of either sex that incremental stimulation of a region homologous to the mammalian substantia nigra pars compacta (SNc) evokes increasing activation of MLR cells with a graded increase in the frequency of locomotor movements. Neurons co-storing glutamate and dopamine were found to project from the primal SNc to the MLR. Blockade of glutamatergic transmission largely diminished MLR cell responses and locomotion. Local blockade of D₁receptors in the MLR decreased locomotor frequency, but did not disrupt the SNc-evoked graded control of locomotion. Our findings revealed the presence of a glutamatergic input to the MLR originating from the primal SNc that evokes graded locomotor movements.SIGNIFICANCE STATEMENTThe mesencephalic locomotor region (MLR) plays a crucial role in the control of locomotion. It projects downward to reticulospinal neurons that in turn activate the spinal locomotor networks. Increasing the intensity of MLR stimulation produces a growing activation of reticulospinal cells and a progressive increase in the speed of locomotor movements. Since the discovery of the MLR some 50 years ago, it has been presumed that higher brain regions activate the MLR in a graded fashion, but this has not been confirmed yet. Here, using a combination of techniques from cell to behavior, we provide evidence of a new glutamatergic pathway activating the MLR in a graded fashion, and consequently evoking a progressive increase in locomotor output.