Cardioinhibitory cardiac vagal neurons (CVNs) do not receive inspiratory-related excitatory inputs under normal conditions. However excitatory purinergic and serotonergic pathways are recruited during inspiratory activity following episodes of hypoxia and hypercapnia (H/H). Prenatal nicotine (PNN) exposure is known to dramatically change cardiorespiratory responses and decrease the ability to resuscitate from H/H. This study tested whether PNN exposure alters excitatory neurotransmission to CVNs in the Nucleus Ambiguus during and following H/H. Spontaneous and inspiratory evoked EPSCs were recorded in CVNs from rats that were exposed to nicotine (6 mg/Kg/day) throughout the prenatal period. In contrast to unexposed animals, in PNN animals H/H recruited excitatory neurotransmission to CVNs during inspiratory-related activity that was blocked by the 34 nicotinic acetylcholine receptor (nAChR) blocker -conotoxin AuIB (-CTX AuIB, 100 µM) and 6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX, 50 µM) and D(-)-2-Amino-5-phosphonopentanoic acid (AP5, 50 µM), selective AMPA/kainate and NMDA receptor blockers, respectively. Following H/H, there was a significant increase in inspiratory-related EPSCs that were unaltered by -CTX AuIB or ondansetron, a 5-HT3 receptor blocker, but were subsequently inhibited by Pyridoxalphosphate-6-azophenyl-2', 4'-disulphonic acid (PPADS, 100 µM), a purinergic receptor blocker and CNQX and AP5. The results from this study demonstrate that with PNN exposure, an excitatory neurotransmission to CVNs is recruited during H/H that is glutamatergic, and dependent upon activation of 34 containing nAChRs. Furthermore, exposure to PNN abolishes a serotonergic long lasting inspiratory-related excitation of CVNs which is replaced by recruitment of a glutamatergic pathway to CVNs post H/H.