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Background:von Willebrand factor (VWF) serves a critical role as a carrier of factor (F)VIII in circulation. While it is generally believed that FVIII and VWF assemble in circulation after secretion from different cells, an alternative view is that cells should exist that co‐express FVIII and VWF.Objectives:In this study, intracellular co‐expression of FVIII and VWF was studied, with particular reference to complex assembly and high‐affinity interaction.Methods:Using yellow fluorescent protein‐tagged FVIII (FVIII‐YFP) and cyan fluorescent protein‐tagged VWF (VWF‐CFP), we studied intracellular trafficking in human embryonic kidney (HEK293) cells and human umbilical vein endothelial cells (HUVEC). The role of the high‐affinity interaction between FVIII and VWF was assessed using a FVIII‐YFP variant carrying a Tyr1680Phe substitution, which abolishes high‐affinity binding to VWF. Cellular trafficking studies were …