Immortalized rat chondrocytes (IRCs) were employed to evaluate the cytocompatibility of different biodegradable polyester scaffolds for chondrocyte seeding and cartilage tissue engineering in vitro due to the limitation of using freshly harvested chondroctyes. Cells were seeded onto the films and the porous substrates as well as into the three-dimensional scaffolds made of the biodegradable polyesters including poly(l-lactide) (PLLA) and two poly(lactide-co-glycolide)s (PLGAs). The materials were characterized by water contact angle, electron spectroscopy for chemical analysis (ESCA), and microscopy. PLGA50/50, one of the PLGAs, had the largest cell numbers at 24 h and 96 h (close to the tissue culture polystyrene control), possibly due to its lower contact angle, higher oxygen/carbon (O/C) atomic ratio, and larger degradation rate. When the surface was further modified by cross-linked Type-II collagen, cell population was significantly enhanced (two- to fourfold). The adhesion and proliferation behavior of IRCs on different materials was parallel to that of rabbit chondrocytes, but was more reproducible in general. IRCs are thus suitable for evaluation of different polymer scaffolds. Despite the favorable cytocompatibility of PLGA50/50, blending with a small portion of PLLA is required for easy fabrication and collagen modification. Scaffolds made of blended materials by freeze-drying procedure with the surface modified by cross-linked Type-II collagen were demonstrated as the ideal templates for chondrocyte seeding in our study.