During early development, GABAergic mechanisms contribute to the regulation of respiratory timing in response to CO2. In 5-7 day old piglets, a double labeling technique was used to determine whether GABA-containing neurons are activated by normoxic hypercapnia (10% CO2, 21% O2, and 69% N2). The c-Fos gene encoded protein (c-Fos) was employed to localize CO2 activated cells within the piglet medulla oblongata. Parvalbumin was used as a marker for GABAergic neurons. In animals breathing room air, only scant c-Fos-like immunoreactive neurons were observed. A marked increase in c-Fos positive cells was induced after a 60 min exposure to hypercapnia. Colocalization studies revealed that hypercapnia significantly increased c-Fos expression in GABA-containing neurons in the medulla oblongata, especially in the ventral aspect of the medulla, within the Bötzinger region, the gigantocellular reticular nucleus, and the caudal raphe nuclei. Only a few double-labeled cells were observed within the nucleus tractus solitarius. Therefore, brainstem GABAergic neurons are part of the neural networks that respond to CO2 and may contribute to respiratory frequency responses to hypercapnia during early development.