Factor V is an essential coagulation cofactor that circulates in plasma and platelet α-granules where it is stored complexed to multimerin 1 (MMRN1). To gain insights into the origin and processing of human platelet factor V, and factor V-MMRN1 complexes, we studied factor V in cultured megakaryocytes. Factor V mRNA was detected in all megakaryocyte cultures. However, like albumin, IgG and fibrinogen, factorV protein was detectable only in megakaryocytes cultured with exogenous protein. The amount of factor V associated with megakaryocytes was influenced by the exogenous factorV concentration. Similar to platelet factor V, megakaryocyte factor V was proteolyzed and complexed with megakaryocyte-synthesized MMRN1. With secretagogues, megakaryocytes released factorV, IgG, fibrinogen and MMRN1. Immunofluorescent and electron microscopy confirmed factorV uptake by endocytosis and its trafficking to megakaryocyte α-granules. These data provide direct evidence that human megakaryocytes process plasma-derived factor V into α-granules and generate factor V-MMRN1 complexes from endogenously and exogenously synthesized proteins.