This study reports a new system that is very effective in identifying genes closely related to the early phase of chondrogenic differentiation. While studying chondrogenesis in a progenitor cell line, ATDC5, we found that the amount of culture media overlying an ATDC5 monolayer affected the extent to which differentiation Occurred. Therefore, to gain insight into the molecular mechanisms of chondrogenic differentiation, differential gene expression between differentiating and non-differentiating ATDC5 cultures was examined by Suppression subtractive hybridization analysis. In this study, we focused on transcription factors that were identified in differentiating cultures, and found that activating transcription factor 5, ATF5, exhibited a conspicuous activation pattern using two methods to induce chondrogenesis of ATDC5 cells. Furthermore, ATF5 was found to be elevated in the developing limb bud by in situ hybridization in a pattern that was highly restricted to the cartilaginous anlagen.. suggesting a positive association with ATF5 expression and chondrogenesis. (c) 2005 Elsevier Inc. All rights reserved.