Genetic strategies for perturbing activity of selected neurons hold great promise for understanding circuitry and behavior. Several such strategies exist, but there has been no direct demonstration of reversible inactivation of mammalian neurons in vivo. We previously reported quickly reversible inactivation of neurons in vitro using expression of the Drosophila allatostatin receptor (AlstR). Here, adeno-associated viral vectors are used to express AlstR in vivo in cortical and thalamic neurons of rats, ferrets, and monkeys. Application of the receptor's ligand, allatostatin (AL), leads to a dramatic reduction in neural activity, including responses of visual neurons to optimized visual stimuli. Additionally, AL eliminates activity in spinal cords of transgenic mice conditionally expressing AlstR. This reduction occurs selectively in AlstR-expressing neurons. Inactivation can be reversed within minutes upon washout of the ligand and is repeatable, demonstrating that the AlstR/AL system is effective for selective, quick, and reversible silencing of mammalian neurons in vivo.