Background/Aims: Exposure to hypoxia triggers a series of responses including hyperventilation and anapyrexia. Hydrogen sulfide (H2S) synthesis is catalyzed by cystathionine β-synthase (CBS) or cystathionine γ-lyase (CSE). The presence and participation of CBS and CSE in response to hypoxia have been described in the carotid body but not yet in brain regions involved in hypoxia-induced hyperventilation and anapyrexia. Therefore, we aimed at investigating the putative role of H2S in ventilatory and thermoregulatory responses to hypoxia. Methods: Surgical procedures were performed under ketamine-xylazine anesthesia (100 and 10 mg/kg, respectively; 1 ml/kg, i.p.). Antibiotics (160.000 U/kg benzylpenicilin, 33.3 mg/kg streptomycin, and 33.3 mg/kg dihydrostreptomycin, i.m.; prophylactically) and analgesic medication (Flunexine; 2.5 mg/kg, s.c.) were provided immediately after the end of surgeries. Male Wistar rats (270-300g, n= 45) were implanted with a guide cannula toward the third ventricle (3V; for microinjection i.c.v.) and with a temperature datalogger capsule in the peritoneal cavity (to record body temperature, Tb). The animals were bolus microinjected into the 3V with a CBS inhibitor (Aminooxyacetate (AOAA), 100 pmol/2 μl, n= 15), or a CSE inhibitor (Propargylglycine (PAG), 160 pmol/2 μl, n= 16) or saline (2 μl, n= 14). After the microinjection, ventilation and Tb of the animals were measured at 5, 10, 20, 30, 40, 50 and 60 minutes in normoxia or hypoxia (7% O2). The results were statistically analyzed by linear mixed model followed by Duncan post hoc test. Results: During normoxia, microinjection of AAOA or PAG did not change ventilation and Tb compared with saline (p>0.05). When microinjected with PAG and exposed to hypoxia, the rats showed a significant increase (p<0.05) in tidal volume (12.57±0.35ml/kg) and ventilation (1674.57±38.24 ml/kg/min) compared with saline (10.90±0.34ml/kg and 1461.78±46.61 ml/kg/min). Interestingly, this increase was even higher when the animals were microinjected with AAOA and exposed to hypoxia, showing a significant increase (p<0,05) in tidal volume (13.90±0,35ml/kg) and ventilation (1971,12±32,57ml/kg/min) not only compared with saline (11,30±0,46ml/kg and 1497,47±31,65ml/kg/min) but also with PAG (12.57±0.35ml/kg and 1674.57±38.24 ml/kg/min). Conclusion: The present results are consistent with the notion that endogenous H2S plays an important role in modulating ventilatory response to hypoxia in rats.