We investigated the behavior of rabbit chondrocytes seeded in vitro into collagen/chitosan/glycosaminoglycan (GAG) scaffolds with different chitosan contents (i.e., at collagen:chitosan ratios of 20 : 1, 5 : 1, and 1.25 : 1, w/w, respectively). The porous scaffolds containing collagen and chitosan were fabricated by using a freeze drying technique and crosslinked using 1-ethyl-3-(3-dimethyl aminopropyl)carbodiimide (EDC) in the presence of chondroitin sulfate (CS). Scanning electron microscope (SEM) views of the scaffolds showed that all three had interconnected pores of mean diameter 164, 353, and 567 ?m at collagen:chitosan ratios of 20 : 1, 5 : 1, and 1 : 1, w/w, respectively. GAG was covalently bound onto these scaffolds at 6.4% (w/w) in all three cases, i.e., regardless of chitosan content. However, increased chitosan content resulted in enhanced mechanical properties and increased pore size. Rabbit chondrocytes seeded onto these scaffolds were cultured for 1, 3, 7, and 14 days. Biochemical analysis of these scaffolds showed that GAG synthesis and proliferation rate increased with time, and this became significant for the collagen : chitosan (20 : 1)-CS scaffold on day 14. The histology of the cell-seeded constructs showed a significantly higher percentage of cells with spherical morphology, consistent with chondrocytic morphology, especially in the collagen : chitosan (20 : 1)-CS scaffold at each time point. This finding was supported by the observation that the pericellular matrix was stained positively for proteoglycans and type II collagen after 14 days. We conclude that the collagen : chitosan (20 : 1)-CS scaffold seems to be a useful carrier material for tissue engineered cartilage.