During the development of de novo synthesized cartilage tissue engineered constructs, transport and biophysical properties are expected to change in time and space. Monitoring and control of the evolution of these parameters are of crucial importance to process biohybrid constructs in vitro. The aim of this work was to measure fluid and macromolecular transport and evolution of mechanical properties of tissue-engineered cartilage constructs as a function of culture time and extracellular matrix (ECM) production. It was found, in agreement with other literature reports, that mechanical and fluid transport properties of the constructs correlated well with time of culture and glycosaminoglycan (GAG) content. Further, diffusion coefficients of 2 probes, dextran (500 kDa) and bovine serum albumin (BSA), correlated well with GAG production. Diffusion coefficients (D) were measured with high spatial and temporal resolution by fluorescent recovery after photobleaching (FRAP). Diffusivity steadily decreases with time while it does not vary through the thickness of the specimen. On the basis of these results, an empirical relationship between diffusion coefficient and GAG content was proposed for the 2 probes analyzed. The results of this study provide useful information to optimize and control the tissue culture process in vitro.