In the present study, we attempted to elucidate mechanisms for the regulation of intracellular calcium levels by H2S in primary rat medullary neurons. Our results showed that NaHS significantly increased the level ofCa2+iin rat medullary neurons in a concentration-dependent manner. L-Cysteine and SAM significantly raised the level ofCa2+iin the medullary neurons while HA and/or AOAA produced a reversal effect. In addition, L-cysteine and SAM significantly increased but HA and/or AOAA decreased the production of H2S in the cultured neurons. TheCa2+ielevation induced by H2S was significantly diminished by EGTA-Ca2+-free solutions, and this elevation was also reduced by nifedipine or nimodipine and mibefradil, suggesting the role of L-type and/or T-type Ca2+channels. Moreover, the effect of H2S onCa2+ilevel in neurons was significantly attenuated by BAPTA-AM and thapsigargin, suggesting the source of Ca2+. Therefore, we concluded that both exogenous and endogenous H2S elevatesCa2+ilevel in primarily cultured rat medullary neurons via both increasing calcium influx and mobilizing intracellular Ca2+stores from ER.