Prosthetic arteriovenous grafts (AVGs) conventionally used for hemodialysis are associated with inferior primary patency rates and increased risk of infection compared with autogenous vein grafts. We tissue-engineered an AVG grown from neonatal human dermal fibroblasts entrapped in bovine fibrin gel that is then decellularized. This graft is both "off-the-shelf" (nonliving) and completely biological. Grafts that are 6 mm in diameter and about 15 cm in length were evaluated in a baboon model of hemodialysis access in an axillary-cephalic or axillarybrachial upper arm AVG construction procedure. Daily antiplatelet therapy was given. Grafts underwent both ultrasound assessment and cannulation at 1, 2, 3, and 6 months and were then explanted for analysis. Excluding grafts with cephalic vein outflow that rapidly clotted during development of the model, 3- and 6-month primary patency rates were 83% (5 of 6) and 60% (3 of 5), respectively. At explant, patent grafts were found to be extensively recellularized (including smoothelin-positive smooth muscle cells with a developing endothelium on the luminal surface). We observed no calcifications, loss of burst strength, or outflow stenosis, which are common failure modes of other graft materials. There was no overt immune response. We thus demonstrate the efficacy of an off-the-shelf AVG that is both acellular and completely biological.