The supply of oxygen within three‐dimensional tissue‐engineered (TE) cartilage polymer constructs is mainly by diffusion. Oxygen consumption by cells results in gradients in the oxygen concentration. The aims of this study were, firstly, to identify the gradients within TE cartilage polymer constructs and, secondly, to predict the profiles during in vitro culture. A glass microelectrode system was adapted and used to penetrate cartilage and TE cartilaginous constructs, yielding reproducible measurements with high spatial resolution. Cartilage polymer constructs were cultured for up to 41 days in vitro. Oxygen concentrations, as low as 2–5%, were measured within the center of these constructs. At the beginning of in vitro culture, the oxygen gradients were steeper in TE constructs in comparison to native tissue. Nevertheless, during the course of culture, oxygen concentrations approached the values measured in native tissue. A mathematical model was developed which yields oxygen profiles within cartilage explants and TE constructs. Model input parameters were assessed, including the diffusion coefficient of cartilage (2.2 × 10⁻⁹) + (0.4 × 10⁻⁹ m² s⁻¹), 70% of the diffusion coefficient of water and the diffusion coefficient of constructs (3.8 × 10⁻¹⁰ m² s⁻¹). The model confirmed that chondrocytes in polymer constructs cultured for 27 days have low oxygen requirements (0.8 × 10⁻¹⁹ mol m⁻³ s⁻¹), even lower than chondrocytes in native cartilage. The ability to measure and predict local oxygen tensions offers new opportunities to obtain more insight in the relation between oxygen tension and chondrogenesis. © 2004 Wiley Periodicals, Inc.