We assessed the invasive capacities and expression of urokinase-type plasminogen activator (uPA) in the estrogen receptor (ER) negative MDA-MB-231 cell line, and the same cell transfected with an ER expression vector (S30 cells) in response to 17 beta-estradiol (E2; 1 nM) and epidermal growth factor (EGF; 1 ng/ml and 10 ng/ml). The invasive potential of S30 cells was only 50% that of MDA-MB-231 cells and was further reduced by E2. EGF increased the invasiveness of S30 cells, but was unable to reverse the inhibitory effects of E2. Invasion of MDA-MB-231 cells was unaffected by E2 or EGF. EGF increased uPA secretion from both cell lines, as determined by ELISA and zymography, and this correlated with increased expression of uPA mRNA. uPA expression in MDA-MB-231 cells was unaffected by E2; however, S30 cells responded to E2 with downregulation of uPA at both the protein and mRNA levels.