The concept of costimulation has been best defined in T cells and B cells. However, other cells that respond in an Ag-specific fashion, such as the mast cell, may be regulated by similar mechanisms. We have found that murine mast cells express one such costimulatory molecule, CD28, which was previously defined as a T and NK cell-specific protein. While CD28 transcription appeared to be constitutive in murine mast cells, its cell surface expression was not. CD28 cell surface expression by mast cells derived from bone marrow with stem cell factor (SCF) was dependent upon activation with agents such as LPS, the Borrelia burgdorferi lipoprotein outer surface protein A, and PMA. Peak cell surface expression of CD28 by such cells occurred 24 h after LPS stimulation, 18 h after outer surface protein A stimulation, and 3 h after PMA stimulation. In contrast, mast cells derived from bone marrow with IL-3 did not demonstrate induction-specific cell surface expression of CD28. Instead, maturation of such cells in vitro allowed for the increased cell surface expression of CD28. Peritoneal mast cells cultured in SCF also expressed CD28. Mast cell CD28 was functional, in that cross-linking of CD28 on the surface of the IL-3-derived cells resulted in an increased level of c-jun transcripts. Additionally, cross-linking of CD28 simultaneously with PMA treatment of SCF-derived mast cells resulted in an increased level of IL-13 transcripts. These data suggest that mast cell CD28 has functions similar to those of T cell CD28.