As assessed by ultrastructure, histochemical staining, and T-cell dependency, in vitro-differentiated inter-leukin 3-dependent mouse mast cells are comparable to the mast cells that reside in the gastrointestinal mucosa but not in the skin or the serosal cavity of the mouse. We now demonstrate that when cloned interleukin 3-dependent mast cells are cocultured with mouse skin-derived 3T3 fibroblasts in the presence of WEHI-3 conditioned medium for 28 days, the mast cells acquire the ability to stain with safranin, increase their histamine content approximately equal to 50-fold and their carboxypeptidase A content approximately equal to 100-fold, and augment approximately equal to 45-fold their biosynthesis of proteoglycans bearing 35S-labeled heparin relative to 35S-labeled chondroitin sulfate glycosaminoglycans. Thus, fibroblasts induce interleukin 3-dependent mouse mast cells to change phenotype from mucosal-like to connective tissue-like, indicating that the biochemical and functional characteristics of this mast cell type are strongly influenced by the connective tissue microenvironment.