Posttranslational histone modifications play important roles in regulating chromatin structure and function. One example of such modifications is histone ubiquitination, which occurs predominately on H2A and H2B. Although the recent identification of the ubiquitin ligase for histone H2A has revealed important roles for H2A ubiquitination in Hox gene silencing as well as in X inactivation, the enzyme(s) involved in H2A deubiquitination and the function of H2A deubiquitination are not known. In order to identify the deubiquitinase for histone H2A, we developed an in vitro deubiquitination assay employing uH2A (ubiquitinated H2A)-containing mononucleosomes as substrates.