We have recently shown that either exogenous or endogenous, transfected OPN induces both uPA expression and increased invasiveness of 21 PT (non-tumorigenic) and 21 NT (tumorigenic) human mammary epithelial cells. Here we asked whether uPA contributes functionally to the increased invasiveness of these cells. The most invasive OPN-transfected cells were assessed for migration through Matrigel in transwell assays, in the presence or absence of various blocking antibodies and uPA inhibitors. Antibodies to both uPA and uPA receptor (uPAR) were shown to significantly inhibit cell invasion, as did the uPA inhibitors (plasminogen activator inhibitor-1 [PAI-1], p-aminobenzamidine [PABN], aprotinin, and amiloride). Both anti-uPA and anti-uPAR antibodies inhibited invasion to a level comparable to that of the control vector transfected cells. In contrast, non-specific IgG showed no antiinvasive effect. Cell migration experiments performed with the parental cell lines in the presence or absence of anti-uPA or anti-uPAR antibodies showed that uPA is also required for migratory responsiveness to exogenous OPN. These data thus provide direct evidence that OPN-induced invasion and migration of these cells requires uPA.