Oxygen is necessary for maintaining cell proliferation and viability and extracellular matrix (ECM) production in 3-dimensional tissue engineering. Typically, diffusion is the primary mode for oxygen transport in vitro; thus, ensuring an adequate oxygen supply is essential. In this study, we determined the oxygen diffusion coefficients of 3 natural ECMs that are being investigated as construct scaffolds for tendon tissue engineering: small-intestine submucosa (SIS), human dermis (Alloderm (R)), and canine fascia lata. Diffusion coefficients were determined using a standard diffusion cell system. The ranges for each matrix type were: SIS: 7 x 10(-6) - 2 x 10(-5) cm(2)/s, Alloderm (R): 1.9 - 3.1 x 10(-5) cm(2)/s, and canine fascia lata: 1.6 - 4 x 10(-5) cm(2)/s. We used the experimental oxygen diffusivity data for these natural ECMs in a mathematical model of oxygen diffusion through a cell-seeded scaffold to estimate the critical size of cell-seeded scaffold that can be cultured in vitro.