A novel method for the preparation of gelatin scaffolds was designed by varying the crosslinking temperature. Four pore size ranges of genipin-crosslinked gelatin scaffolds were made by varying the crosslinking temperature from 10 to 25 degrees C, with the pore sizes ranging from 50 to 500 microm. The pore size of the scaffold increases as the crosslinking temperature increases. Articular chondrocytes of Wistar rats were in vitro cultured in these scaffolds. DNA assay, glycosaminoglycan (GAG) assay, hematoxylin-eosin staining, Safranin-O staining and reverse transcription-polymerase chain reaction were performed to analyze the effect of the pore size on cell growth and the secretion of extracellular matrix (ECM). As the pores become larger, the rate of cell growth and the amount of GAG secretion increase, and the expressions of all four gene markers for aggrecan, collagen type I, collagen type II and collagen type X increase. The cells in the smaller pores often show a dedifferentiated form. The phenotype of the cells is maintained better in larger pores. Chondrocytes prefer the group of scaffolds with pore size between 250 and 500 microm for better proliferation and ECM production. The size of the space for cell growth is a key factor for cell metabolism.