The cholinergic agonist carbachol was conjugated to latex microspheres that were fluorescently labeled with rhodamine and used as neuroanatomical probes that show little diffusion from their injection site and retrogradely label neurons projecting to the injection site. Microinjection of this pharmacologically active probe into the gigantocellular field of the cat pontine brain stem caused the awake cats to fall into rapid movement (REM) sleep indistinguishable from that produced by free carbachol. Three-dimensional computer reconstruction of the retrogradely labeled neurons revealed a widely distributed neuronal network in the pontine tegmentum. These pharmacologically active microspheres permit a new precision in the characterization and mapping of neurons associated with the control of behavioral state and of other cholinergic networks.