The purpose of this study was to compare the effect of different hydrogels on the production of tissue‐engineered cartilage based on polyglycolic acid (PGA). Chondrocytes were isolated from adult sheep auricles. Alginate, Type I collagen, methylcellulose, and pluronic F127 hydrogels were evaluated, as were controls prepared without hydrogels. Proliferated chondrocytes were mixed with each hydrogel at 20 × 10 ⁶ cells/mL and seeded onto PGA (1 × 1 × 0.2 cm, n = 60). The constructs were cultured with serum‐free medium containing 5 ng/mL TGF‐β ₂ and 5 ng/mL des(1‐3)IGF‐I in rotational bioreactors for up to 6 weeks. The cellular morphology, histology, and biochemistry were analyzed. Type I collagen, methylcellulose, and pluronic F127 displayed improved cartilage matrix deposition in terms of histology and biochemistry compared to alginate. It was not concluded that the combined seeding of chondrocytes and hydrogels on a PGA scaffold had significantly better effects than cell seeding without hydrogels. However, the histology and other useful findings in this ECM analyses suggested that Type I collagen and MC hydrogels were the best candidates for cartilage regeneration, because of their stimulation for chondrocyte proliferation in a three‐dimensional culture as well as cartilage regeneration. © 2005 Wiley Periodicals, Inc. J Biomed Mater Res, 2005