Hydrogen sulfide (H2S) is endogenously produced in the brain from L-cysteine by the enzyme cystathionine beta-synthase (CBS) and functions as a neuromodulator in the brain. H2S selectively enhances NMDA receptor-mediated responses and alters hippocampal long-term potentiation (LTP). The production of H2S is regulated by Ca2+/calmodulin-mediated pathways and is enhanced in response to neuronal excitation. In addition to this fast regulation, we describe here a slower form of the regulation of H2S production by testosterone and S-adenosyl-L-methionine (SAM), a CBS activator. Endogenous H2S in the mouse brain increases after birth, reaches a maximum level at 8 weeks and then decreases. Female brain contains less H2S than male brain at each age. A single administration of testosterone to female mice increases the endogenous H2S and SAM, which reach levels similar to those of male mice. In contrast, castration of male mice decreases the levels of testosterone, SAM and H2S in the brain. Administration of SAM once a day for 3 days increases the brain H2S without significantly changing the testosterone level. These observations suggest that testosterone can regulate the brain H2S level via changing the level of SAM.