The present study describes the morphological assessment of chondrogenic potency during a cell expanding process through serial subculturing of rabbit chondrocytes at different levels of population doublings; (PD) in a T-flask with a conventional polystyrene surface. The passaged populations were seeded on a high-density collagen surface (CL surface) and in a collagen gel (CL gel) scaffold to evaluate the planar and spatial morphologies of the chondrocytes, respectively, as well as the gene expressions of mRNA for collagen types I and II. The planar morphological estimation was based on roundness (R-c) of chondrocyte cells at different PD values after I day incubation on the CL surface. The frequency of round-shaped cells with R-c > 0.9 (f(R)) decreased with increasing PD values, accompanied by an increase in collagen type I mRNA level. At PD = 17.8, the frequency reached f(R) = 0.12, which was less than one-sixth of that at PD = 0. A similar trend was found with respect to the passaged chondrocytes embedded in the CL gels by estimating the spatial morphology in terms of sphericity (S-c) determined 4 days after seeding. With an increase in PD value, the frequency in spherical-shaped cells with S-c > 0.9 (fs) decreased and the mRNA expression of collagen type I increased, giving f(s) = 0.28 at PD = 17.8 which was less than a quarter of that at PD = 0. From these results, the cell morphologies on the CL surface and in the CL gel were proposed as indicators for understanding chondrogenic potentials concerning the phenotypes and differentiated states in the population during cell expansion, ultimately leading to quality control of tissue-engineered cartilage. (c) 2009, The Society for Biotechnology, Japan. All rights reserved.