Selective inhibition of the Na(+)/H(+) exchanger type 3 (NHE3) increases the firing rate of brainstem ventrolateral CO(2)/H(+) sensitive neurons, resembling the responses evoked by hypercapnic stimuli. In anesthetized animals, NHE3 inhibition has also been shown to stimulate the central chemosensitive drive. We aimed to analyze the respiratory-related brainstem regions affected by NHE3 inhibition in anaesthetized spontaneously-breathing rats with intact peripheral afferents. For that, c-Fos immunopositive cells were counted along the brainstem in rats intravenously infused with the selective NHE3 inhibitor AVE1599. A rostral extension of the ventral respiratory column which includes the pre-Bötzinger complex was activated by the NHE3 inhibitor. In addition, the number of c-Fos positive cells resulted significantly increased in the most rostral extension of the retrotrapezoid nucleus/parapyramidal region. In the pons, the intravenous infusion of AVE1599 activated the lateral parabrachial and Kölliker-Fuse nuclei. Thus, selective NHE3 inhibition in anaesthetized rats activates the respiratory network and evokes a pattern of c-Fos expressing cells similar to that induced by hypercapnia.