In endochondral ossification, chondrogenesis precedes bone development. Cartilage differentiation is initiated by the formation of chondrogenic cell condensates. Thus, it is essential to investigate genes expressed at the condensation stage for a better understanding of chondrogenesis and ossification. To this end, we constructed a cDNA library from the mRNA fraction derived from a chondrogenic cell line (ATDC5) at the cell condensation stage by using the signal sequence trap by retrovirus-mediated expression (SST-REX) method. We obtained 486 factor (IL-3)-independent clones by screening 5.7 x 10(3) clones. DNA sequencing analysis of the clones identified genes encoding 157 known proteins and 4 novel proteins. These 4 genes encoding novel proteins were expressed not only in chondrogenic ATDC5 cells but also in osteogenic MC3T3-E1 and myogenic C2C12 cells. The mRNA expression level of 1 of the 4 clones increased at the calcification stage of the differentiation of MC3T3-E1 cells. The results demonstrate that the SST-REX method is a useful experimental system to identify genes involved in the complicated mechanisms of bone formation.