The effect of the addition of monosaccharides composing glycosaminoglycans (GAGs) on the accumulation of type 2 collagen (COL(II)) in a three-dimensional (3D) culture of porcine chondrocyte cells was investigated for possible application to cartilage regenerative medicine. Primary chondrocytes from porcine cartilage were cultivated in three-dimension employing atelo collagen gel for 3 weeks with the addition of several saccharides. The addition Of D-glucuronic acid (D-GlcA), N-acetyl-D-galactosamine (D-GalNAc), chondroitin sulfate C (CSC), D-galactose, N-acetyl-D-glucosamine, and L-iduronic acid increased markedly not aggrecan but COL(II) accumulation although the addition of D-fructose and D-mannose not composing GAGs did not show such an effect. The addition of D-GlcA and D-GalNAc had no synergistic effect. The addition of CSC, D-GlcA, and D-GalNAc also increased COL(II) mRNA expression while aggrecan mRNA expression was not increased by these compounds. In conclusion, the addition of monosaccharides composing GAGs might be valuable for increasing COL(II) accumulation in the 3D culture of chondrocytes. (C) 2009, The Society for Biotechnology, Japan. All rights reserved.