Factor IX (FIX) binds to collagen IV (Col4) in the subendothelial basement membrane. In hemophilia B, this FIX-Col4 interaction reduces the plasma recovery of infused FIX and plays a role in hemostasis. Studies examining the recovery of infused BeneFix (FIXWT) in null (cross-reactive material negative, CRM2) hemophilia B mice suggest the concentration of Col4 readily available for binding FIX is ∼405 nM with a 95% confidence interval of 374 to 436 nM. Thus, the vascular cache of FIX bound to Col4 isseveral-fold the FIX level measured inplasma. In a mouse model of prophylactic therapy (testing hemostasis by saphenous vein bleeding 7 days after infusion of 150 IU/kg FIX), FIXWT and the increased half-life FIXs Alprolix (FIXFC) and Idelvion (FIXAlb) produce comparable hemostatic results in CRM2 mice. In bleeding CRM2hemophilia B mice, the times to first clot at a saphenous vein injury site after the infusions of the FIX agents are significantly different, at FIXWT < FIXFC < FIXAlb. Dysfunctional forms of FIX, however, circulate in the majority of patients with hemophilia B (CRM1). In the mouse prophylactic therapy model, none of the FIX products improves hemostasis in CRM1 mice expressing a dysfunctional FIX, FIXR333Q, that nevertheless competes with infused FIX for Col4 binding and potentially other processes involving FIX. The resultsinthis mouse model of CRM1hemophiliaB demonstrate that the endogenous expression of a dysfunctional FIX can deleteriously affect the hemostatic response to prophylactic therapy.