The Integral membrane protein 2A (Itm2A) is a transmembrane protein belonging to a family composed of at least two other members, Itm2B and Itm2C, all of them having a different expression pattern. The Itm2a gene serves as a marker for early stages in endochondral ossification. In order to understand the role of Itm2A in this process, expression of the gene was investigated in different cell systems. In C3H10T1/2 cells, the gene was upregulated early on when the cells were induced to the chondrogenic lineage but less to the osteogenic lineage. In MCT cells, expression was upregulated at permissive temperatures but not at non-permissive temperatures. When induced with insulin, ATDC5 cells expressed Itm2a in early stages but not at late stages. Furthermore, PTH treatment seems to upregulate Itm2a transcription. In order to understand the role of Itm2a in the chondrogenic differentiation process in more detail, we constitutively overexpressed exogenous Itm2A in mouse ATDC5 cells. Two clones expressing high levels of Itm2a were isolated and characterized. Gene expression analysis of the overexpresser clones demonstrated that expression of collagen type X was delayed. These results demonstrate that overexpression of Itm2a in mouse ATDC5 cells impede the transition to hypertrophic cells. Taken together, our observation supports the involvement of Itm2a in the early stages of chondrogenesis in vitro.