Human cytomegalovirus was shown to bind to human umbilical vein endothelial cells in a specific, saturable and calcium-dependent manner (Kd = 7.9 pM (4 degrees C), 6469 virus binding sites/cell). Affinity adsorption of detergent-prepared lysates of surface-radiolabeled endothelial cells to virions resulted in the identification of cell-derived proteins of approximate M(r) 36,000 and 32,000 that bound cytomegalovirus. Protein sequencing of peptides obtained by cyanogen bromide cleavage demonstrated that the 36 kDa protein corresponded to human annexin II, and the 32 kDa protein was likely a degradation product. Purified annexin II was demonstrated to bind directly to virions (Kd = 57 nM, 688 annexin II binding sites/virion). These results provide evidence that an endothelial cell-surface form of annexin II acts as a receptor for cytomegalovirus, and indicate a previously undescribed role for annexin II.