Posttranslational modifications of histones play an important role in the regulation of chromatin structure and, hence, gene regulation. Recently, we have identified a novel modification of histones: binding of the vitamin biotin to lysine residues in histones H2A, H3, and H4. Here, we describe a procedure to identify those amino acids that are targets for biotinylation in histones. Briefly, the following analytical sequence is used to identify biotinylation sites: (i) short peptides (<20 amino acids in length) are synthesized chemically; amino acid sequences in the peptides are based on the sequence in a given region of a given histone; (ii) peptides are incubated with biotinidase or holocarboxylase synthetase to conduct enzymatic biotinylation; and (iii) biotin in peptides are probed using streptavidin peroxidase. Amino acid substitutions (e.g., lysine-to-alanine substitutions) in synthetic peptides can be used to corroborate identification of biotinylation sites.