Nucleus isthmi (NI) is a mesencephalic structure of the amphibian brain that has been reported to participate in CO2 chemoreception and in the ventilatory response to hypoxia. However, no information exists about the modulators and/or mediators involved. In the present study, we assessed the participation of nitric oxide (NO) in the hypoxic and hypercarbic drive to breathing, specifically in the NI. We compared the ventilatory and cardiovascular responses with hypoxia and hypercarbia after microinjecting 100 nmol/0.5 μl of N G-nitro-l-arginine methyl ester (l-NAME; an NO synthase blocker) into the NI of toads ( Bufo paracnemis). l-NAME had no effect under resting conditions. Hypoxia elicited an increase in ventilation in control and vehicle toads by elevating tidal volume (VT). Hypercarbia caused hyperventilation in all groups due to an increase in both VT and frequency. The microinjection ofl-NAME into the NI elicited an increase in ventilatory response to hypoxia and hypercarbia due to a higher VT. We conclude that NO in the NI has an inhibitory effect when the respiratory drive is high, acting on VT.