AbstractDiffusion is likely to be the primary mechanism for macromolecular transport in tissue‐engineered cartilage, and providing an adequate nutrient supply via diffusion may be necessary for cell proliferation and extracellular matrix production. The goal of this study was to measure the diffusivity of tissue‐engineered cartilage constructs as a function of scaffold material, culture conditions, and time in culture. Diffusion coefficients of four different‐sized fluorescent dextrans were measured by fluorescence recovery after photobleaching in tissue‐engineered cartilage constructs seeded with human adipose‐derived stem cells or acellular constructs on scaffolds of alginate, agarose, gelatin, or fibrin that were cultured for 1 or 28 days in either chondrogenic or control conditions. Diffusivities in the constructs were much greater than those of native cartilage. The diffusivity of acellular constructs increased 62% from Day 1 to Day 28, whereas diffusivity of cellular constructs decreased 42% and 27% in chondrogenic and control cultures, respectively. The decrease in diffusivity in cellular constructs is likely due to new matrix synthesis, which may be enhanced with chondrogenic media, and matrix contraction by the cells in the fibrin and gelatin scaffolds. The increase in diffusivity in the acellular constructs is probably due to scaffold degradation and swelling. © 2004 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 70B: 397–406, 2004