Factor V is endocytosed by megakaryocytes from plasma to form the platelet-derived factor V/Va pool via a receptor-mediated, clathrin-dependent mechanism. However, the megakaryocyte receptor that mediates the binding and subsequent endocytosis of plasma-derived factor V is unknown. Because of its known ability to interact with proteins involved in coagulation and fibrinolysis, the role of low-density lipoprotein receptor-related protein (LRP) or an LRP-like molecule was examined in factor V endocytosis by the megakaryocyte-like cell line CMK. As endocytosis by such proteins is Ca2+-dependent, binding and endocytosis of factor V ± added Ca2+ were examined. While endocytosis of factor V was absolutely dependent upon the presence of Ca2+, binding of factor V to megakaryocytes was unaffected by its absence allowing for the quantification of 125I-factor V binding in the absence of factor V endocytosis. The time-dependent, specific 125I-factor V binding to megakaryocytes reached a steady-state within 20–25 min and displayed a sigmoidal character. The steady state binding of a plasma concentration of 125I-factor V could be displaced 56.4 ± 7.8% by the addition of a 150-fold molar excess of the LRP ligand, receptor associated protein (RAP). In contrast, nearly all of the 125I-factor V bound could be displaced following the addition of a 50-fold molar excess of factor V alone or by the addition of both factor V and RAP. These observations suggest that factor V binds to two binding sites on megakaryocytes, which consist of a specific factor V receptor and LRP (or an LRP-like molecule). This notion of a two-receptor system was confirmed in steady state, concentration-dependent binding analyses of 125I-factor V in the presence or absence of RAP. Binding of factor V was saturable at a concentration ~15–20 times higher than the plasma concentration of factor V, which is consistent with observations that the platelet-derived factor V concentration is dependent upon and parallels the plasma-derived factor V concentration. Similar to the observations described above, concentration-dependent binding was also inhibited 40–50% by the presence of excess RAP. The binding curves in the presence or absence of RAP remained sigmoidal and were fit with confidence (r ≥ 0.95) to a two-receptor binding model. In support of these observations, AlexaFluor488-labeled factor V and AlexaFluor633-labeled RAP co-localized within the same cells subsequent to their endocytosis by megakaryocytes as demonstrated by confocal microscopy. Flow cytometric analyses of the same cell population confirmed these observations: All of the cells that endocytosed factor V also endocytosed RAP. Based upon the combined observations we propose a binding model where factor V endocytosis is mediated by a two-receptor system consisting of a specific factor V receptor and an LRP co-receptor closely linked on the cell surface. In this model, factor V binds to its specific receptor in a Ca2+-independent manner. Bound factor V is then transferred to LRP (or an LRP-like molecule) to allow for the binding of a second factor V molecule to the unoccupied site on the factor V receptor. Factor V bound to LRP is then endocytosed in Ca2+- and clathrin-dependent manners. As our flow cytometric analyses indicate that all of the cells bind and endocytose RAP, we hypothesize that it is the presence or absence of the specific factor V receptor that defines the megakaryocytes’ ability to endocytose factor V from plasma.