We have previously described thrombin (Th)-protease nexin 1 (PN1) inhibitory complex binding to cell surface heparins and subsequent low density lipid receptor-related protein (LRP)-mediated internalization. Our present studies examine the catabolism of urinary plasminogen activator (uPA)-PN1 inhibitory complexes, which, unlike Th.PN1 complexes, bind almost exclusively through the uPA receptor. In addition, the binding site in PN1 required for the LRP-mediated internalization of Th.PN1 complexes is not required for the LRP-mediated internalization of uPA.PN1 complexes. Thus, the protease moiety of the complex partially determines the mechanistic route of entry. Because cell surface heparins are only minimally involved in the binding and internalization of uPA.PN1 complexes, we then predicted that complexes between uPA and the heparin binding-deficient PN1 variant, PN1(K7E), should be catabolized at the same rate as complexes formed with native PN1. Surprisingly, the uPA.PN1(K7E) complexes were degraded at only a fraction of the rate of native complexes. Internalization studies revealed that both uPA. PN1(K7E) and native uPA.PN1 complexes were initially internalized at the same rate, but uPA.PN1(K7E) complexes were rapidly retro-endocytosed in an intact form. By examining the pH dependence of complex binding in the range of 4.0-7.0, it was determined that the uPA.PN1 inhibitory complexes must specifically bind to endosomal heparins at pH 5.5 to be retained and sorted to lysosomes. These studies are the first to document a role for heparins in the catabolism of SERPIN-protease complexes at a point further in the pathway than cell surface binding, and this role may extend to other heparin-binding LRP-internalized ligands.