B7-H1 is a recently described B7-like molecule that costimulates T-cell growth and cytokine secretion without binding to CD28, cytotoxic T-lymphocyte antigen-4 (CTLA-4), and inducible costimulator (ICOS). In this report, a mouse homologue of human B7-H1 is identified, and its immunologic functions are studied in vitro and in vivo. Mouse B7-H1 shares 69% amino acid homology to the human counterpart. Similar to human B7-H1, mouse B7-H1 can be induced to express on macrophages, T cells, and B cells and to enhance T-cell proliferation and secretion of interleukin-10 (IL-10), interferon-γ, and granulocyte-macrophage colony-stimulating factor but not IL-2 and IL-4. Furthermore, B7-H1 preferentially costimulates CD4⁺ T cells independently of CD28 and enhances mixed lymphocyte responses to allogeneic antigens. In contrast to B7-1, expression of B7-H1 on murine P815 tumor cells by transfection fails to increase allogeneic and syngeneic cytolytic T-cell responses in vitro and in vivo. Administration of B7-H1Ig fusion protein, however, enhances keyhole limpet hemocyanin– specific T-cell proliferation and 2,4,6-trinitrophenyl–specific immunoglobulin G2a antibody production. The study thus identifies a unique costimulatory pathway that preferentially affects T-helper cell functions.