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The covalent binding of biotin to histones participates in heterochromatin formation, cell cycle progression and cellular response to DNA breaks. Biotinylation of histones appears to be a reversible process, but the identity of enzymes that remove biotin marks is largely unknown. Our long-term goal is to identify histone debiotinylases in human cells. Here we developed an avidin-based plate assay to quantify histone debiotinylase activities in nuclear extracts. This assay is an essential first step in purifying and identifying histone debiotinylases from human cells. Using this assay, we demonstrated that debiotinylation of histones depends on temperature and pH, consistent with enzyme catalysis. Experiments with purified histones, proteases and protease inhibitors provide evidence that removal of biotin marks from histones is mediated by debiotinylases rather than by proteases. Activities of histone debiotinylases varied among human tissues: colon=lung>placenta=liver>lymphoid cells. This assay proved useful in monitoring activities of putative histone debiotinylases during their partial purification from cells. Collectively, this assay is a useful tool for investigating histone debiotinylases in human tissues.