The generation of a cartilage substitute will require both the mechanical and biochemical properties of the substance to approach that of native tissue. Recent studies (Buschmann et al., 1992, 1995; Bader and Lee, 1997) have shown that agarose can provide a suitable environment for the production of a mechanically functional matrix. Over time cells seeded in this matrix alter the intrinsic mechanical properties of the matrix. Additionally, several studies have included such biochemical factors as vitamin C (ascorbate) to the culture medium. This biological effector has varied effects on chondrocyte matrix biosynthesis. Specifically, it has been shown to promote calcification in chick growth plate chondrocytes (Wu et al., 1989), while it increased both type II and type I procollagen mRNA in 5 day culture (Sandell and Daniel, 1988). Conversely, it has been shown in bovine culture that aggrecan mRNA levels rise steadily in monolayer culture without ascorbate. but rise quickly and then fall off in its presence (Hering et al., 1994). To assess the effect of ascorbate in our system, primary bovine articular chondrocytes seeded in agarose, we initiated a study to examine the effects of ascorbate on both the production of cartilage extracellular matrix and the development of mechanical properties over a 14 day culture period. Three groups were studied: agarose disks without cells (control) and cell-seeded agarose disks maintained in DMEM supplemented daily with and without 50 μg/ml of ascorbate. Glycosaminoglycan (GAG) and hydroxyproline contents of each disk were determined using standard colorimetric assays.