Post-translational modifications of histones play an important role in regulating chromatin dynamics and function. One such modification, methylation, is involved in the regulation of the epigenetic program of a cell, determining chromatin structure, and regulating transcription. Methylation of histones occurs on both lysine and arginine residues, and until recently, was thought to be an irreversible process. The recent discovery of histone demethylases revealed that histone methylation is more dynamic than previously recognized. The October issue of Cold Spring Harbor Protocols features a set of methods from Keiichi Nakayama and colleagues from Kyushu University for investigating demethylase activity. The protocol, In Vitro Histone Demethylase Assay, describes two different in vitro histone demethylase enzyme reactions and three different methods for measuring histone demethylase activity. These methods can be applied to measuring histone demethylase activity in tissues and cell lysates, identification of novel histone demethylases, and screening for inhibitors of histone demethylases. As one of our featured articles, the protocol is freely available to subscribers and nonsubscribers alike.