Nicotine, acting at nicotinic acetylcholine receptors (nAChRs), is the primary addictive component of tobacco. Smokers often report an anxiolytic effect of cigarettes. This relief of anxiety, attributed to nicotine, is an important contributor to relapse when smokers try to quit. Hence, the study of the anxiolytic effects of nicotine is important for understanding the mechanisms underlying nicotine addiction. Mammalian nAChRs are pentameric ion channels usually composed ofα andβ subunits. Taking advantage ofβ4-homozygous-null mice (β4-/-), we examined the role of the nAChR β4 subunit in anxiety-related behaviors. The β4-/- mice behaved as though they were less anxious than wild-type littermates on the elevated-plus and staircase mazes, tests that measure anxiety-related behaviors. To obtain an independent, physiological indication of the stress produced by several tests, we measured changes in heart rate using telemetry. Consistently with the behavioral phenotype, β4-/- mice had a smaller heart rate increase in the elevated-plus maze than did wild-type littermates. In contrast, during social isolation, a separate test for anxiety,β4-/- mice exhibited a greater increase in heart rate than did wild-type littermates. Finally, β4-/- mice were indistinguishable from their wild-type littermates in the open field, the light/dark box, and the mirrored chamber. The overall results demonstrate that β4-containing (β4*) nAChRs influence behavioral responses during anxiety-related tests, and that this effect depends on the type of anxiety-provoking experience. Through their influence on anxiety-related behavior, β4* nAChRs might influence both tobacco consumption and smoking relapse.