Dopamine transmission is critical for exploratory motor behavior. A key regulator is acetylcholine: forebrain acetylcholine regulates striatal dopamine release, whereas brainstem cholinergic inputs regulate the transition from tonic to burst firing modes of dopamine neurons. How these sources of cholinergic activity combine to control dopamine efflux and exploratory motor behavior is unclear. Here we show that mice lacking total forebrain acetylcholine exhibit enhanced frequency dependent striatal dopamine release and are hyperactive in a novel environment, whereas mice lacking rostral brainstem acetylcholine are hypoactive. Exploratory motor behavior is normalized by removal of both cholinergic sources. Involvement of dopamine in the exploratory motor phenotypes observed in these mutants is indicated by their altered sensitivity to the dopamine D2 receptor antagonist raclopride. These results support a model in which forebrain and brainstem cholinergic systems act in tandem to regulate striatal dopamine signaling for proper control of motor activity.