A high density collagen type I coated substrate (CL substrate) was used to evaluate the chondrocyte phenotypes in passaged cultures. With increasing age of cell population (population doubling (PD) = 0-14.5), the frequency of non-dividing spindle shaped cells without ALP activity increased, accompanied with an increase in gene expression of collagen type I. meaning the senescence of dedifferentiated cells. At the middle age of cell population (PD = 5.1 and 6.6), the high frequency of polygonal shaped cells with ALP activity existed on the CL substrate together with up-regulated expressions of collagen types II and X, indicating the terminal differentiation of chondrocytes. When the chondrocytes passaged up to the middle age were embedded in collagen gel, the high frequency of single hypertrophic cells with collagen type II formation was recognized, which supports the thought that the high gene expression of collagen type II was attributed to terminal differentiation rather than redifferentiation. These results show that the CL substrate can draw out the potential of terminal differentiation in chondrocytes, which is unattainable by a polystyrene surface, and that the CL substrate can be a tool to evaluate cell quality in three-dimensional culture with the collagen gel. (C) 2011, The Society for Biotechnology, Japan. All rights reserved.