Surprisingly, a successful human pregnancy requires cells from the fetal portion of the placenta (cytotrophoblasts) to adopt tumor-like properties. Cytotrophoblasts attach the conceptus to the endometrium by invading the uterus, and they initiate blood flow to the placenta by breaching maternal vessels. But unlike tumor metastasis, cytotrophoblast invasion is highly regulated both spatially and temporally. Our previous work showed that matrix metalloproteinase-9 (MMP-9) expression is upregulated during cytotrophoblast differentiation along the invasive pathway, and that activity of this proteinase specifies the cells' ability to degrade extracellular matrix (ECM) substrates in vitro. Here we tested the hypothesis that invading cytotrophoblasts express an unusual tissue inhibitor of metalloproteinase (TIMP) repertoire that allows them to regulate their MMP-9 proteolytic activity. By using protease-substrate gel electrophoresis, we found that human cytotrophoblasts express primarily TIMP-3. We showed that the cells' TIMP-3 expression is regulated in accord with that of MMP-9. The highest levels of protein and mRNA for both these molecules were detected after differentiation to a fully invasive phenotype and during early gestation, when invasion peaks, rather than at term, when invasion has stopped. Our results suggest that coexpression of MMP-9 and TIMP-3 by invading cytotrophoblasts plays an important role in regulating the depth of uterine invasion.