Mast cells are well known for their harmful role in IgE-mediated hypersensitivity reactions, but their physiological role remains a mystery. Several recent studies have reported that mast cells play a critical role in innate immunity in mice by releasing tumor necrosis factor α (TNF-α) to recruit neutrophils to sites of enterobacterial infection. In some cases, the mast cell TNF-α response was triggered when these cells directly bound FimH on the surface of Escherichia coli. We have identified CD48, a glycosylphosphatidylinositol-anchored molecule, to be the complementary FimH-binding moiety in rodent mast cell membrane fractions. We showed that (i) pretreatment of mast cell membranes with antibodies to CD48 or phospholipase C inhibited binding of FimH⁺ E. coli, (ii) FimH⁺ E. coli but not a FimH⁻ derivative bound isolated CD48 in a mannose-inhibitable manner, (iii) binding of FimH⁺ bacteria to Chinese hamster ovary (CHO) cells was markedly increased when these cells were transfected with CD48 cDNA, and (iv) antibodies to CD48 specifically blocked the mast cell TNF-α response to FimH⁺ E. coli. Thus, CD48 is a functionally relevant microbial receptor on mast cells that plays a role in triggering inflammation.