Magnocellular regions of the basal forebrain contain cholinergic neurons that project to the cerebral cortex. Neurons in the same basal forebrain regions innervate the brainstem. The present study investigated whether these brainstem projecting neurons are cholinergic, project also to the cortex, and share similar physiological properties as cortically projecting neurons. Data with retrograde tracing from various regions of the pons, medulla, and cortex combined with choline acetyltransferase immunofluorescence indicated that: 1) brainstem projecting neurons are usually segregated from cortically projecting and/or cholinergic neurons in the basal forebrain, 2) virtually no brainstem projecting neurons in the basal forebrain are cholinergic, and 3) only rarely do basal forebrain neurons have axon collaterals that project to both cortex and brainstem. Extracellular recordings from basal forebrain neurons confirmed the paucity of axonal collateralization and the topographic segregation between cortically and brainstem projecting basal forebrain neurons, and, in addition, showed that brainstem projecting neurons have a slower mean conduction velocity than cortically projecting neurons. These observations suggest that basal forebrain neurons projecting to the brainstem (pons, medulla) and the cortex represent separate cell populations in terms of projections, neurotransmitter content, distribution, and physiological properties.