OBJECTIVES: There is significant room for improvement in the development of tissue-engineered blood vessels (TEBVs) for vascular reconstruction. Most commonly, TEBVs are seeded with endothelial cells (ECs) only. This provides an antithrombogenic surface but suboptimal physiologic characteristics compared with native arteries, due to lack of smooth muscle cells (SMCs) in the vessel media. Although SMCs are critical in vessel architecture and function throughout the vascular tree, few studies have incorporated SMCs in TEBVs implanted in vivo. As such, the goal of the present study was to evaluate the effect of SMC coseeding with ECs on TEBV maturation, structure, and function after prolonged in vivo maturation. METHODS: Dual-seeded TEBVs (dsTEBVs) were created by coseeding autologous ECs derived from circulating progenitor cells and SMCs from artery explants onto the lumen and outer surface of extracellular matrix scaffolds, respectively. Control vessels were seeded with ECs alone (ecTEBV). All vessels were preconditioned to pulsatile flow for 10 to 14 days in a bioreactor, implanted as arterial interposition grafts in sheep, and allowed to heal and adapt in vivo for 4 months before ex vivo physiologic testing and histologic analysis. RESULTS: All implants were patent at 4 months. There were no structural failures, aneurysms, or infectious complications. The dsTEBVs exhibited a greater degree of wall maturation, characterized by higher medial cellularity (P = .01) and greater percentage of α-actin (P = .005) and SMC-specific muscle myosin heavy chain (P = .005) staining compared with ecTEBVs. Contractile responses to phenylephrine and serotonin were significantly greater in isolated rings of dsTEBVs than those observed in ecTEBVs (P = .01). CONCLUSIONS: To our knowledge, this is the first study that demonstrates enhanced in vivo wall maturation and contractile function of TEBVs coseeded with autologous SMCs and ECs compared with EC seeding alone. These data suggest a coseeding strategy can be accomplished in a clinically relevant timeframe (typically 6 weeks) and may provide advantages for arterial reconstruction compared with vessels engineered only with endothelium. CLINICAL RELEVANCE: Bioengineering of functional blood vessels using autologous cells has been proposed as an alternative to prosthetic grafts for patients who lack suitable vein. Previous studies have focused on attaching endothelial cells to the luminal wall of prosthetic or tissue-engineered vessels. However, vascular smooth muscle cells (SMCs), which comprise the bulk of the artery wall, are essential in regulating artery wall geometry, tone, and integrity in response to a variety of hemodynamic and biochemical stimuli. Therefore, the addition of an SMC layer to an engineered blood vessel would enhance its function. This study evaluated the effect of incorporating vascular SMCs and endothelium into an engineered blood vessel that was implanted for 4 months in vivo. All vessels remained patent and maintained stable wall geometry without significant stenosis, dilation, or rupture. Dual-seeded vessels demonstrated enhanced structure and function with greater numbers of cells expressing SMC markers in the medial layer and significantly improved vasomotor responses than did vessels seeded with endothelium alone. This strategy may soon evolve into a clinically useful alternative for patients lacking vein for vascular reconstructions.