The outcome of human pregnancy depends on the differentiation of cytotrophoblasts, specialized placental cells that physically connect the embryo/fetus to the mother. As cytotrophoblasts differentiate, they acquire tumor-like characteristics that enable them to invade the uterus. In a novel feedback loop, the increasingly higher levels of oxygen they encounter within the uterine wall influence their differentiation into vascular-like cells. Together, the invasive and cell surface properties of cytotrophoblasts enable them to form vascular connections with uterine blood vessels that divert maternal blood flow to the placenta, a critical hurdle in pregnancy. It is therefore important to understand how cytotrophoblasts respond to changes in oxygen tension. Here we used a proteomics approach, two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) combined with mass spectrometry, to characterize the protein repertoire of first trimester human cytotrophoblasts that were maintained under standard tissue culture conditions (20% O(2)). 2-D PAGE showed a unique protein map as compared to placental fibroblasts and human JEG-3 choriocarcinoma cells. Mass spectrometry allowed the identification of 43 spots on the cytotrophoblast map. Enzymes involved in glycolysis and responses to oxidative stress, as well as the 14-3-3 signaling/adapter proteins, were particularly abundant. Hypoxia in vitro (2% O(2)) produced discrete changes in the expression of a subset of proteins in all the aforementioned functional categories. Together, these data offer new information about the early gestation cytotrophoblast protein repertoire and the generalized mechanisms the cells use to respond to changes in oxygen tension at the maternal-fetal interface.