Neuronal nicotinic acetylcholine receptors (nAChR) are present in high abundance in the nervous system (Decker et al., 1995). There are a large number of subunits expressed in the brain that combine to form multimeric functional receptors. We have generated an alpha(4) nAChR subunit knock-out line and focus on defining the behavioral role of this receptor subunit. Homozygous mutant mice (Mt) are normal in size, fertility, and home-cage behavior. Spontaneous unconditioned motor behavior revealed an ethogram characterized by significant increases in several topographies of exploratory behavior in Mt relative to wild-type mice (Wt) over the course of habituation to a novel environment. Furthermore, the behavior of Mt in the elevated plus-maze assay was consistent with increased basal levels of anxiety. In response to nicotine, Wt exhibited early reductions in a number of behavioral topographies, under both unhabituated and habituated conditions; conversely, heightened levels of behavioral topographies in Mt were reduced by nicotine in the late phase of the unhabituated condition. Ligand autoradiography confirmed the lack of high-affinity binding to radiolabeled nicotine, cytisine, and epibatidine in the thalamus, cortex, and caudate putamen, although binding to a number of discrete nuclei remained. The study confirms the pivotal role played by the alpha(4) nAChR subunit in the modulation of a number of constituents of the normal mouse ethogram and in anxiety as assessed using the plus-maze. Furthermore, the response of Mt to nicotine administration suggests that persistent nicotine binding sites in the habenulo-interpeduncular system are sufficient to modulate motor activity in actively exploring mice.