The serine protease uPA (urokinase-type plasminogen activator) and its receptor uPAR (CD87) are often elevated in malignant tumours, hence, inhibition of this tumour-associated plasminogen activation system provides an attractive target for therapeutic strategies. WX-UK1, a derivative of 3-aminophenylalanine in the L-conformation with inhibitory antiproteolytic properties, was tested for its specificity spectrum using specific chromogenic paranitroanilide peptide substrates. The corresponding D-enantiomer of WX-UK1 was used as a control. The anti-tumour and anti-metastatic (number of lung foci and weight of the axillary lymph nodes) properties were studied by subcutaneous administration of WX-UK1 to Brown Norwegian (BN) rats carrying orthotopically transplanted BN472 rat breast tumours. WX-UK1 selectively inhibited tumour-related proteases from rats and humans such as uPA, plasmin, or thrombin in the sub or low micromolar range. The activity was stereoselective as the D-enantiomer of WX-UK1 inhibited uPA and plasmin at approximately 70-fold higher Ki values than the active L-form. Chronical administration of the L-enantiomer of WXUK1 impaired primary tumour growth and metastasis of BN472 rat breast cancer in a dose-dependent manner. The minimum inhibitory dosage with maximal effect was between 0.15 and 0.3 mg/kg/day. The inactive D-enatiomer of WX-UK1 was not active in this respect. Daily treatment with WX-UK1 for up to 35 days was well tolerated as judged by the unchanged body and organ weight development. In conclusion, our results provide evidence that WX-UK1 as a single agent inhibits breast tumour growth and metastasis in vivo, and thus is a promising candidate drug to treat human cancer.