The authors have established a permanent human cell line, EAhy 926, that expresses factor VIII-related antigen (VIII:Ag), a highly differentiated function of vascular endothelium. This cell line was derived by fusing human umbilical vein endothelial cells (HUV-EC) with the permanent human cell line A549. The hybrid cells, which had more chromosomes than either progenitor cell type, maintained VIII:Ag expression for over 100 cumulative population doublings, including more than 30 passages and three cloning steps. The expression of VIII:Ag in these hybrid cells was confirmed by immunofluorescence and accumulation in the culture fluid. The morphology and distribution of VIII:Ag in EAhy 926 cells are indistinguishable from those in primary endothelial cells. The study suggests that intraspecies hybrids can sustain the expression of differentiated functions, and the use of rhodamine 6G pretreatment may enhance the yield of viable hybrids. This cell line provides a valuable tool for in vitro studies and research on hemostasis and related processes.The authors have established a permanent human cell line, EAhy 926, that expresses factor VIII-related antigen (VIII:Ag), a highly differentiated function of vascular endothelium. This cell line was derived by fusing human umbilical vein endothelial cells (HUV-EC) with the permanent human cell line A549. The hybrid cells, which had more chromosomes than either progenitor cell type, maintained VIII:Ag expression for over 100 cumulative population doublings, including more than 30 passages and three cloning steps. The expression of VIII:Ag in these hybrid cells was confirmed by immunofluorescence and accumulation in the culture fluid. The morphology and distribution of VIII:Ag in EAhy 926 cells are indistinguishable from those in primary endothelial cells. The study suggests that intraspecies hybrids can sustain the expression of differentiated functions, and the use of rhodamine 6G pretreatment may enhance the yield of viable hybrids. This cell line provides a valuable tool for in vitro studies and research on hemostasis and related processes.