Both elevated homocysteine and decreased hydrogen sulfide (H(2)S) are observed in the brains of Alzheimer's disease (AD) patients. Reactive oxygen species (ROS) overproduction contributes to the neurotoxicity of homocysteine; however, H(2)S is an endogenous antioxidant gas. Therefore, the aim of this study was to investigate whether the imbalance of proportion to this endogenous protective antioxidant gas is involved in homocysteine-caused neurotoxicity. We show that homocysteine inhibits the generation of endogenous H(2)S and the expression and activity of cystathionine-β-synthetase (CBS), the main enzyme responsible for the generation of H(2)S in PC12 cells. S-Adenosylmethionine, an activator of CBS, not only prevents homocysteine-induced inhibition of endogenous H(2)S production but also attenuates homocysteine-triggered cytotoxicity and accumulation of ROS. We find that activation of ERK1/2 occurs in homocysteine-treated PC12 cells and blockade of ERK1/2 with U0126 abolished the homocysteine-induced cytotoxicity and inhibitory effect on endogenous H(2)S generation. These results indicate that homocysteine neurotoxicity involves reduction of H(2)S production, which is caused by inhibition of CBS and mediated by activation of ERK1/2. Our study suggests a promising future of H(2)S-based therapies for neurodegenerative diseases such as AD.