The rat retrotrapezoid nucleus (RTN) contains pH-sensitive neurons that are putative central chemoreceptors. Here, we examined whether these neurons respond to peripheral chemoreceptor stimulation and whether the input is direct from the solitary tract nucleus (NTS) or indirect via the respiratory network. A dense neuronal projection from commissural NTS (commNTS) to RTN was revealed using the anterograde tracer biotinylated dextran amine (BDA). Within RTN, 51% of BDA-labelled axonal varicosities contained detectable levels of vesicular glutamate transporter-2 (VGLUT2) but only 5% contained glutamic acid decarboxylase-67 (GAD67). Awake rats were exposed to hypoxia (n = 6) or normoxia (n = 5) 1 week after injection of the retrograde tracer cholera toxin B (CTB) into RTN. Hypoxia-activated neurons were identified by the presence of Fos-immunoreactive nuclei. CommNTS neurons immunoreactive for both Fos and CTB were found only in hypoxia-treated rats. VGLUT2 mRNA was detected in 92 +/- 13% of these neurons whereas only 12 +/- 9% contained GAD67 mRNA. In urethane-chloralose-anaesthetized rats, bilateral inhibition of the RTN with muscimol eliminated the phrenic nerve discharge (PND) at rest, during hyperoxic hypercapnia (10% CO(2)), and during peripheral chemoreceptor stimulation (hypoxia and/or i.v. sodium cyanide, NaCN). RTN CO(2)-activated neurons were recorded extracellularly in anaesthetized intact or vagotomized rats. These neurons were strongly activated by hypoxia (10-15% O(2); 30 s) or by NaCN. Hypoxia and NaCN were ineffective in rats with carotid chemoreceptor denervation. Bilateral injection of muscimol into the ventral respiratory column 1.5 mm caudal to RTN eliminated PND and the respiratory modulation of RTN neurons. Muscimol did not change the threshold and sensitivity of RTN neurons to hyperoxic hypercapnia nor their activation by peripheral chemoreceptor stimulation. In conclusion, RTN neurons respond to brain P(CO(2)) presumably via their intrinsic chemosensitivity and to carotid chemoreceptor activation via a direct glutamatergic pathway from commNTS that bypasses the respiratory network. RTN neurons probably contribute a portion of the chemical drive to breathe.