The pivotal role of mast cells in allergic reactions and inflammatory processes is well established and recent studies have suggested that mast cells may also have a role in specific immune responses. Because mast cells have been shown to phagocytose and kill enterobacteria, we wished to determine whether they could also process bacterial Ags for presentation to T cells. Using a model system in which a well-characterized T cell epitope is expressed within bacteria as a fusion protein, we demonstrate in this paper that mast cells are indeed capable of processing bacterial Ags for presentation through class I MHC molecules to T cell hybridomas after phagocytic uptake of live bacteria. Processing occurs from a number of Gram-negative enterobacteria including Salmonella typhimurium and Escherichia coli. Parallel assays show that processing of the model Ag from enterobacteria by mast cells is similar in efficiency to processing by peritoneal macrophages. Consistent with earlier observations demonstrating a function of the bacterial fimbrial protein FimH in promoting bacterial binding to mast cells, the magnitude of the Ag processing response of E. coli is influenced by bacterial expression of FimH. Taken together, these observations extend the range of cell types capable of the phagocytic pathway of Ag processing and suggest that mast cells may have a previously unrecognized role in the induction of specific immune responses to bacteria.