Butyrophilin (BTN) genes encode a set of related proteins. Studies in mice have shown that one of these, BTN1A1, is required for milk lipid secretion in lactation, whereas butyrophilin-like 2 is a coinhibitor of T cell activation. To understand these disparate roles of BTNs, we first compared the expression and functions of mouse Btn1a1 and Btn2a2. Btn1a1 transcripts were not restricted to lactating mammary tissue but were also found in virgin mammary tissue and, interestingly, spleen and thymus. In confirmation of this, BTN1A1 protein was detected in thymic epithelial cells. By contrast, Btn2a2 transcripts and protein were broadly expressed. Cell surface BTN2A2 protein, such as the B7 family molecule programmed death ligand 1, was upregulated upon activation of T cells. We next examined the potential of both BTN1A1 and BTN2A2 to interact with T cells. Recombinant Fc fusion proteins of murine BTN2A2 and, surprisingly BTN1A1, bound to activated T cells, suggesting the presence of one or more receptors on these cells. Immobilized BTN-Fc fusion proteins, but not MOG-Fc protein, inhibited the proliferation of CD4 and CD8 T cells activated by anti-CD3. BTN1A1 and BTN2A2 also inhibited T cell metabolism, IL-2, and IFN-gamma secretion. Inhibition of proliferation was not abrogated by exogenous IL-2 but could be overcome following costimulation with high levels of anti-CD28 Ab. These data are consistent with a coinhibitory role for mouse BTNs, including BTN1A1, the BTN expressed in the lactating mammary gland and on milk lipid droplets.