T cell activation and immune function are regulated by costimulatory molecules of the B7 superfamily. Human B7-H3 is a recent addition to this family and has been shown to mediate T cell proliferation and IFN-gamma production. In this work we describe the identification of the mouse B7-H3 homolog, which is ubiquitously expressed in a variety of tissues. Activated CD4 and CD8 T cells express a putative receptor that can be recognized by soluble mouse B7-H3-Ig molecules. While the mouse B7-H3 gene was found to contain a single copy, we discovered a novel isoform of human B7-H3 (named as B7-H3b hereafter) with four Ig-like domains that results from gene duplication and differential splicing. B7-H3b is the major isoform expressed in several tissues. This structural information suggests a genetic variation of the B7-H3 gene in mammalian species.