Acetylcholine profoundly affects neocortical function, being involved in arousal, attention, learning, memory, sensory and motor function, and plasticity. The majority of cholinergic afferents to neocortex are from neurons in nucleus basalis. Nucleus basalis also contains projecting neurons that release other transmitters, including GABA and possibly glutamate. Hence, electrical stimulation of nucleus basalis evokes the release of a mixture of neurotransmitters in neocortex, and this lack of selectivity has impeded research on cholinergic signaling in neocortex. We describe a method for the selective stimulation of cholinergic axons in neocortex. We used the Cre-lox system and a viral vector to express the light-activated protein channelrhodopsin-2 in cholinergic neurons in nucleus basalis and their axons in neocortex. Labeled neurons depolarized on illumination with blue light but were otherwise unchanged. In anesthetized mice, illumination of neocortex desynchronized the local field potential, indicating that light evoked release of ACh. This novel technique will enable many new studies of the cellular, network, and behavioral physiology of ACh in neocortex.