Background: Collagen-rich tendons and ligaments are important for joint stability and force transmission, but the capacity to form new tendon is poorly understood. In the present study, we investigated mechanical strength, fibril size, and structure during development of tendon-like tissue from adult human tenocytes (termed tendon constructs) in vitro over 5 weeks in 3D tissue culture. Results: The constructs displayed large elongated tendon cells aligned along the tendon axis together with collagen fibrils that increased in diameter by 50% from day 14 to 35, which approaches that observed in adult human tendon in vivo. The increase in diameter was accompanied by a 5-fold increase in mechanical strength (0.9±0.1 MPa to 4.9±0.6 MPa) and Young's modulus (5.8±0.9 MPa to 32.3±4.2 MPa), while the maximal strain at failure (16%) remained constant throughout the 5-week culture period. Conclusions: The present study demonstrates that 3D tendon constructs can be formed by isolated human tendon fibroblasts, and when these constructs are subjected to static self-generated tension, the fibrils will grow in size and strength approaching that of adult human tendon in vivo. Developmental Dynamics 242:2–8, 2013. © 2012 Wiley Periodicals, Inc.