Low concentrations of inhaled hydrogen sulfide (H 2S) induce hypometabolism in mice. Biological effects of H 2S in in vitro systems are augmented by lowering O 2 tension. Based on this, we hypothesized that reduced O 2 tension would increase H 2S-mediated hypometabolism in vivo. To test this, male Sprague-Dawley rats were exposed to 80 ppm H 2S at 21% O 2 or 10.5% O 2 for 6 h followed by 1 h recovery at room air. Rats exposed to H 2S in 10.5% O 2 had significantly decreased body temperature and respiration compared with preexposure levels. Heart rate was decreased by H 2S administered under both O 2 levels and did not return to preexposure levels after 1 h recovery. Inhaled H 2S caused epithelial exfoliation in the lungs and increased plasma creatine kinase-MB activity. The effect of inhaled H 2S on prosurvival signaling was also measured in heart and liver. H 2S in 21% O 2 increased Akt-P Ser473 and GSK-3β-P Ser9 in the heart whereas phosphorylation was decreased by H 2S in 10.5% O 2, indicating O 2 dependence in regulating cardiac signaling pathways. Inhaled H 2S and low O 2 had no effect on liver Akt. In summary, we found that lower O 2 was needed for H 2S-dependent hypometabolism in rats compared with previous findings in mice. This highlights the possibility of species differences in physiological responses to H 2S. Inhaled H 2S exposure also caused tissue injury to the lung and heart, which raises concerns about the therapeutic safety of inhaled H 2S. In conclusion, these findings demonstrate the importance of O 2 in influencing physiological and signaling effects of H 2S in mammalian systems. © 2012 the American Physiological Society.