Cyclooxygenase-2 (COX-2) increases breast cancer cell invasion. Expression of various pro-angiogenic and pro-invasive factors has been correlated with high expression of COX-2. However, whether these factors are essential to COX-2-mediated breast cancer invasion, and the mechanisms by which COX-2 increases the expression of these factors are unknown. Our microarray results indicate that higher COX-2 expression was associated with increased levels of interleukin-8 (IL-8), a key factor in breast cancer invasion and metastasis. COX-2 overexpressing cells (MCF-7/COX-2), generated by transfecting COX-2-encoding plasmids into the poorly invasive MCF-7 breast cancer cells, were more invasive and produced higher IL-8 levels than the parental cells. To investigate the role of IL-8 in COX-2-mediated invasion, MCF-7 parental cells were incubated with IL-8. Exogenous IL-8 increased the invasiveness of MCF-7 cells. IL-8 is one pathway by which COX-2 mediates breast cancer invasion. Protein kinase A (PKA) and protein kinase C (PKC) are activated by COX-2 and are involved in IL-8 regulation. Inhibition of PKC, not PKA, decreased IL-8 production and invasion in MCF-7/COX-2 cells. Activation of PKC, not PKA, increased IL-8 production and invasion in MCF-7 cells. Thus, the invasive effects of COX-2 are mediated by PKC, not PKA. Activity of the urokinase-type plasminogen activator (uPA) was increased in MCF-7 cells by COX-2 overexpression or by the addition of a PKC activator or by IL-8. Inhibition of PKC decreased uPA activity in MCF-7/COX-2 cells. Furthermore, inhibition of uPA activity decreased the invasiveness of MCF-7/COX-2 cells, indicating that uPA was essential to COX-2-mediated invasion. Herein we demonstrate for the first time a detailed mechanism by which COX-2 increases breast cancer invasion: the PKC/IL-8/uPA pathway.