The role of endogenously produced H2S in mediating varied physiological effects in mammals has spurred enormous recent interest in understanding its biology and in exploiting its pharmacological potential. In these early days in the field of H2S signaling, large gaps exist in our understanding of its biological targets, its mechanisms of action and the regulation of its biogenesis and its clearance. Two branches within the sulfur metabolic pathway contribute to H2S production: (i) the reverse transsulfuration pathway in which two pyridoxal 5'-phosphate-dependent (PLP) enzymes, cystathionine beta-synthase and cystathionine gamma-lyase convert homocysteine successively to cystathionine and cysteine and (ii) a branch of the cysteine catabolic pathway which converts cysteine to mercaptopyruvate via a PLP-dependent cysteine aminotransferase and subsequently, to mercaptopyruvate sulfur transferase-bound persulfide from which H2S can be liberated. In this review, we present an overview of the kinetics of the H2S-generating reactions, compare the structures of the PLP-enzymes involved in its biogenesis and discuss strategies for their regulation. This article is part of a Special Issue entitled: Pyridoxal Phospate Enzymology. (C) 2011 Elsevier B.V. All rights reserved.