The effect of endogenous dopamine on the activity of target neurons recorded with patch clamp or Ca²⁺ imaging techniques in slices has been studied to date with intra-striatal stimuli. Yet, this approach is severely handicapped by the nonphysiological and nonspecific stimulation of local neurons and fibers within the striatum. We now report a new juvenile and adult mouse slice preparation in which a component of the nigro-striatal dopaminergic pathway is preserved in its entirety, from cell bodies to axon terminals. This tilted parasagittal slice (380–400 μm) just medial to the subthalamic nucleus contains functional nigro-striatal neurons as assessed by morphological examination of tyrosine hydroxylase positive cell bodies and axons, combined with electrochemical assays of dopamine release in the striatum in response to stimulation of the substantia nigra pars compacta. The nigro-striatal slice constitutes a suitable in vitro preparation to determine the impact of endogenously released dopamine on target neurons of the striatum.