To examine whether AP-2' is a critical component of the genetic program that directs human trophoblast differentiation, we used DNA microarray analyses to characterize the effects of a dominant negative form of the AP-2 protein upon in vitro differentiating cytotrophoblast cells. Human cytotrophoblast cells (>95% pure) were cultured for 3 days in the presence of control medium or medium containing an adenovirus that expresses a dominant-negative mutant of AP-2 (Ad2.AP-2D/N) or an adenovirus lacking the AP-2 mutant gene (Ad.WT). DNA microarray analyses using Affymetrix human U95Av2 GeneChips were performed on RNA extracted from the three groups of cells immediately prior to and after three days of cell culture. Cells infected with Ad2.AP-2D/N or Ad2.WT underwent morphological differentiation similar to that of uninfected cells, with greater than 90% of the cells in each group fusing to form multinucleated syncytiotrophoblast cells. However, Ad2.AP-2D/N markedly inhibited the induction or repression of many genes that were regulated in the non-infected and Ad2.WTinfected cells during differentiation. Eighteen of the 25 most induced genes and 17 of the 20 most repressed genes during differentiation were AP-2-dependent with the majority of these related to extracellular organization, cellular communication and signal transduction. Taken together, these findings strongly suggest that AP-2 plays a critical role for both the induction and repression of genes that comprise post-syncytialization gene expression programs of trophoblast differentiation and maturation. AP-2, however, is not required for the fusion of cytotrophoblast cells to form a syncytium or the expression of syncytin.