The urokinase receptor (uPAR) is linked to plasma membranes through a glycosylphosphatidylinositol (GPI) anchor. It has been posited that the GPI anchor facilitates clearance of uPAR-bound complexes between two chain urokinase (tcuPA) and plasminogen activator inhibitor type 1 (PAI-1) by the alpha 2-macroglobulin receptor (alpha 2MR) which permits re-expression of unoccupied uPA receptors on the cell surface. To test this hypothesis we compared internalization and degradation of 125I-labeled tcuPA-PAI-1 by COS cells expressing either transfected wild-type, GPI-linked uPAR (uPAR/GPI), or a chimeric receptor composed of the extracellular domains of uPAR linked to the transmembrane and cytosolic domains of the alpha chain (p55 subunit) of the interleukin-2 receptor (uPAR/IL-2R alpha). The kinetics of binding, internalization and degradation of tcuPA-PAI-1 by COS cells expressing each form of uPAR were virtually identical. However, internalization of complexes by uPAR/IL-2R alpha was more susceptible to inhibition by recombinant soluble 39-kDa alpha 2MR-associated protein (RAP) which competes for binding of tcuPA-PAI-1 complexes to alpha 2MR (p < 0.001), and the internalization was accompanied by a greater reduction in the number of surface uPAR/IL-2R alpha, than uPAR/GPI (p < 0.05). These studies indicate that the rate of internalization of tcuPA-PAI-1 is governed primarily by the extracellular domains of uPAR, whereas the GPI anchor may facilitate internalization of complexes and re-expression of uPAR when binding sites on alpha 2MR are limiting.