Carbachol when injected into the basal forebrain alters spontaneous motor behavior and usually decreases locomotion. However, the extent of the brain area producing this effect has not yet been determined. The goal of the present study was to use quantitative mapping of injection sites to further localize the effect of carbachol on spontaneous locomotion of rats. The distance travelled by an animal and the time spent moving were simultaneously measured before and after injection of carbachol or saline into 96 sites in the basal forebrain. Each site was injected with 1.0 microgram (5.47 nmol) of carbachol, a dose close to ED50, in a volume of 0.2 microliter. A decrease in spontaneous locomotion was obtained as a result of injections of carbachol into the preoptic and anterior hypothalamic areas, particularly into the medial preoptic nucleus and the latero-anterior hypothalamic nucleus. The area from which a consistent decrease in spontaneous locomotion was obtained was surrounded by an area producing an increase in locomotion with a narrow zone of overlap. This decrease in locomotion was dose dependent and reversed by atropine. The results indicate that both the decreasing and increasing effects of carbachol on locomotion are anatomically specific and that the decreasing effects can be elicited from a limited forebrain area. It is suggested that muscarinic cholinergic mechanisms in the basal forebrain may be involved in the pathogenesis of neural dysfunction associated with locomotor activity in man.