The interaction of T cell CD28/CTLA-4 receptors with B7-1 activation Ag on APC represents an important costimulatory pathway in T cell activation. However, it is now evident that this costimulatory pathway is neither unique nor universal for the activation of T cells. Our previous study indicated that a 60-kDa membrane protein, recognized by mAb 2D10, was expressed before B7 by activated murine B cells. This molecule was critically involved in activation of T cells in response to auto- and alloantigens. In the present study, we report on the isolation of a cDNA for this early T cell costimulatory molecule (ETC-1). ETC-1, like B7-1, is a member of the Ig supergene family and is composed of 303 amino acids. Nucleic acid sequence comparison indicated that ETC-1 is identical to the B7-2 molecule. When expressed in Chinese hamster ovary cells, ETC-1 showed profound T cell costimulatory activity as demonstrated by its ability to enhance CD4 T cell proliferation in response to Con A or anti-CD3 stimulation. Furthermore, ETC-1 also bound to both CD28-Ig and CTLA4-Ig fusion proteins. These results strongly support the notion that the interaction of ETC-1/B7-2 with CD28 or CTLA-4 receptors represents an alternative T cell costimulatory pathway.