Prenatal nicotine exposure alters breathing and ventilatory responses to stress through stimulation of nicotine acetylcholine receptors (nAChRs). We tested the hypothesis that α4-containing nAChRs are involved in mediating the effects of prenatal nicotine exposure on ventilatory and metabolic responses to intermittent mild asphyxia (MA). Using open-flow plethysmography, we measured ventilation (V˙E) and rate of O2 consumption (V˙O2) of wild-type (WT) and α4-knock-out (KO) mice, at postnatal (P) days 1-2 and 7-8, with and without prenatal nicotine exposure (6mgkg-1day-1 beginning on embryonic day 14). Mice were exposed to seven 2min cycles of mild asphyxia (10% O2 and 5% CO2), each interspersed with 2min of air. Compared to WT, α4 KO mice had increased air V˙E and V˙O2 at P7-8, but not P1-2. Irrespective of age, genotype had no effect on the hyperventilatory response (increase in V˙E/V˙O2) to MA. At P1-2, nicotine suppressed air V˙E and V˙O2 in both genotypes but did not affect the hyperventilatory response to MA. At P7-8 nicotine suppressed air V˙E and V˙O2 of only α4 KO's but also significantly enhanced V˙E during MA (nearly double that of WT; p<0.001). This study has revealed complex effects of α4 nAChR deficiency and prenatal nicotine exposure on ventilatory and metabolic interactions and responses to stress.