This article reviews the chemistry, measurement, metabolism, and pharmacokinetics of the cytoprotective agent amifostine. Validated analytic methodology to measure parent drug and pharmacologically active metabolites and pharmacokinetic studies are essential to the efficient performance and analysis of clinical studies. Well-validated analytic methods developed in the authors' laboratory were used to characterize this agent. Amifostine [s-2-(3-aminopropylamino)ethyl-phosphorothioate] is the phosphorylated pro-drug form of the active free thiol drug WR-1065 [2-(3-aminopropylamino)ethanethiol]. Observations described here support the hypothesis that amifostine is dephosphorylated rapidly by alkaline phosphatase present on the plasma membranes of the arteriolar endothelium of various normal tissues and on the plasma membranes of the kidneys' proximal tubular epithelium to its active thiol metabolite WR-1065, which in turn immediately enters normal tissues. Other metabolites that have been identified are WR-33278, the symmetrical disulfide of WR-1065; the mixed disulfides WR-1065-cysteine and WR-1065-glutathione; and cysteamine. Amifostine was recently approved by the US Food and Drug Administration for use as a cytoprotector in cancer patients receiving chemotherapy. Current pharmacokinetic studies in cancer patients are focusing on establishing a population model as a basis for developing limited sampling strategies for future investigations of the pharmacokinetic-pharmacodynamic behavior of amifostine.