Mild reductions in ambient temperature dramatically increase the mortality of neonatal mice deficient in pituitary adenylate cyclase-activating polypeptide (PACAP), with the majority of animals succumbing in the 2nd postnatal week. During anesthesia-induced hypothermia, PACAP^-/- mice at this age are also sensitive to prolonged apneas and sudden death. From these observations, we hypothesized that before the onset of genotype-specific mortality and in the absence of anesthetic, the breathing of PACAP-deficient mice is more susceptible to mild reductions in ambient temperature than wild-type littermates. To test this hypothesis, we recorded breathing in one group of postnatal day 4 (P4) PACAP^+/+, ^+/- and ^-/- neonates (using unrestrained, flow-through plethysmography) and metabolic rate in a separate group (using indirect calorimetry), both of which were exposed acutely to ambient temperatures slightly below (29°C), slightly above (36°C), or at thermoneutrality (32°C). At 32°C, the breathing frequency of PACAP^-/- neonates was significantly less than PACAP^+/+ littermates. Reducing the ambient temperature to 29°C caused a significant suppression of tidal volume and ventilation in both PACAP^+/- and ^-/- animals, while the tidal volume and ventilation of PACAP^+/+ animals remained unchanged. Genotype had no effect on the ventilatory responses to ambient warming. At all three ambient temperatures, genotype had no influence on oxygen consumption or body temperature. These results suggest that during mild reductions in ambient temperature, PACAP is vital for the preservation of neonatal tidal volume and ventilation, but not for metabolic rate or body temperature.