We show that maintenance but not initiation of engrailed (en) gene expression in the Drosophila embryo requires trithorax (trx), which is also required to maintain stable long-term expression of the homeotic genes throughout the development. Like the homeotic genes, en expression is dependent on trx in only a subset of embryonic cells normally expressing en, including specific cells in the nervous system and the dorsal fat body cells surrounding the gonad. Loss of en expression in the dorsal fat body is correlated with the sterility of en females which also carry trx mutations. In addition, trx is required for normal en expression in the posterior compartment of the developing wing, reflected in enhancement of en phenotypes in en adults which also carry trx mutations. trx appears to be dispensable for maintenance of en expression in other embryonic cells. The trx protein binds to the region of the polytene chromosomes which contains the en gene, suggesting that trx regulates en expression directly by binding to the en regulatory region.