Nicotinic receptors containing the 4 subunit (4* nAChRs) have high sensitivity and are widely expressed in the CNS, yet their contributions to behavioral tolerance, a hallmark of nicotine dependence, are unclear. To evaluate the contribution of 4*- and non-4 nAChRs in the development of tolerance to hypothermia and locomotor suppression, 4 knockout (KO), hypersensitive Leu9'Ala 4 knock-in, and wild-type (WT) mice received daily nicotine injections, and their behaviors were compared. Repeated selective activation of 4* nAChRs in Leu9'Ala mice produced profound tolerance to hypothermia over 7 days, whereas no tolerance was observed in 4 KO animals. The summed time course and temperature response (after appropriate normalizations) from these two mutant mouse strains resembled the time course of WT tolerance. In addition, daily selective activation of 4* nAChRs elicited locomotor activation in Leu9'Ala mice, but nicotine suppressed activity in 4 KO mice and this did not change with daily drug exposure. Again, appropriately combined responses from the two mutant strains resembled the biphasic nicotine-induced activity in WT animals. Thus, by analyzing nicotinic responses in two complementary mouse lines, one lacking 4* nAChRs, the other expressing hypersensitive 4* nAChRs, one can accurately separate non-4 nAChR responses from 4 nAChR responses, and one can also account for WT tolerance to both hypothermia and locomotor suppression. Our study suggests a new paradigm for bridging the gap between genetic manipulation of a single receptor and whole-animal behavioral studies, and shows that activation of 4* nAChRs is both necessary and sufficient for the expression of tolerance.