Previous studies have shown that several proteolytic enzymes are associated with membrane protrusions at the leading edge of migrating tumor cells. In this study we demonstrate that seprase and the urokinase plasminogen activator receptor (uPAR), co-localize in the plasma membrane of LOX malignant melanoma cells. Cells were labeled with fluorochrome-conjugated monoclonal antibodies (mAb) directed against seprase and uPAR. Proximity between these two molecules was detected with resonance energy transfer (RET) imaging, single-cell emission spectrophotometry, and single-cell excitation spectrophotometry. Significant RET signals were detected on LOX cells when adherent to uncoated and extracellular matrix (ECM)-coated surfaces. This indicates that seprase and uPAR are within approximately 7 nm in the plasma membrane of LOX cells. When LOX cells adhered to a 3D extracellular-like matrix, seprase-uPAR complexes were found to be associated with invadopodia. Further microscopy experiments demonstrated gelatinolytic activity, a functional attribute of seprase, in association with seprase-uPAR membrane domains. Formation of seprase-uPAR membrane complexes is dependent upon both the cytoskeleton and integrins. Specifically, the involvement of beta(1)-integrins was demonstrated by the inhibition of RET by an inhibitory anti-beta(1)-integrin mAb. Based on these findings, we speculate that formation of heterogeneous lytic domains in the invading membranes of LOX cells increases the efficiency of directed pericellular proteolysis.