Nicotine, the main addictive component of tobacco, evokes a wide range of dose-dependent behaviors in rodents, and when administrated in high doses, it can induce clonic-tonic seizures. Nicotine acts through the nicotinic acetylcholine receptors (nAChRs). Mutations in the human α4- and the β2-nAChR subunit genes cause autosomal dominant nocturnal frontal lobe epilepsy. Using transgenic mice with mutations in nAChR subunits, it was demonstrated previously that the α4-, α5-, and α7-subunits are involved in nicotine-induced seizures. To examine the possibility that the β4-subunit is also involved in this phenotype, we tested mice with homozygous β4-subunit deficiency. The β4 null mice were remarkably resistant to nicotine-induced seizures compared with wild-type and α5 null mice. We also generated mice with double deficiency of both α5- and β4-nAChR subunits and demonstrated that they were more resistant to nicotine’s convulsant effect than either the α5 or the β4 single mutant mice. In addition, the single α5 mutants and the double α5β4-deficient mice exhibited a significantly shorter latency time to seizure than that of the wild-type mice. Our results thus show that β4-containing nAChRs have a crucial role in the pathogenesis of nicotine-induced seizures. Furthermore, by comparing multiple mutant mice with single and double subunit deficiency, we suggest that nicotinic receptors containing either α5- or β4-subunits are involved in nicotine-induced seizures and that receptors containing both subunits are likely to contribute to this phenomena as well. However, the α5-subunit, but not the β4-subunit, regulates the rate of response to high doses of nicotine.