Hydrogen sulfide (H2S) is a gasotransmitter synthesized from cysteine (Cys) by pyridoxal-5′-phosphate-dependent enzymes. We investigated the potential roles of H2S in the regulation of central rhythmic respiration in adult rats in vivo. Sodium hydrosulfide (NaHS: 2.5 mM, 10 mM, and 5 mM) as a source of exogenous H2S, Cys (2.5 mM, 10 mM and 5 mM) as a source of endogenous H2S, 2.5 mM Cys+10 mM hydroxylamine (NH2OH), and 10 mM NH2OH, respectively, were intracerebroventricularly injected into rats. The rhythmic discharge of the diaphragm, including burst duration (BD), burst interval (BI), burst frequency (BF), and integrated amplitude (IA), and arterial blood pressure (BP) were measured at different time points. The results were analyzed by analysis of variance. A total of 2.5 mM NaHS did not significantly affect changes in BD, BI, BF, IA, or BP (P>0.05), whereas 2.5 mM Cys significantly altered BD, BI, and BF (P<0.05); however, there was no change in IA and BP (P>0.05). A concentration of 5 mM Cys had effects similar to those of 5 mM NaHS; both induced biphasic respiratory responses and changed the BF (P<0.05). A concentration of 10 mM NH2OH irreversibly inhibited rhythmic discharge of the diaphragm except for IA. No change was seen in BI, BF, IA, or BP (P>0.05) except for BD was temporarily decreased (P<0.05) in the 2.5 mM Cys+10 mM NH2OH group. These results suggest that exogenous and endogenous H2S may participate in the regulation of respiratory activity in adult rats.