Activation of Hox gene clusters is an early event in embryonic development since individual members play important roles in patterning of the body axis. Their functions require precise control of spatiotemporal expression to provide positional information for the cells of the developing embryo, and the manner by which this control is achieved has generated considerable interest. The situation is different in pluripotent cells, where HOX genes are not expressed but are held in potentio as bivalent chromatin domains, which are resolved upon differentiation to permit HOX cluster activation. In this study we have used differentiation of the pluripotent embryonal carcinoma cell line NTera2SP12 and the human embryonic stem cell line H9 to examine epigenetic changes that accompany activation of the HOXA cluster and show that specific genomic loci are marked by lysine methylation of histone H3 (H3K4 tri- and dimethyl, H3K9 trimethyl) and acetylation of histone H4 even in the undifferentiated cells. The precise locations of such modified histones may be involved in controlling the colinear expression of genes from the cluster.