Study of the role of mast cells in the human gastrointestinal tract has suffered from the inability to examine these cells in vitro. In addition, work in rodent systems suggests that there are substantial differences between intestinal mast cells and those from other tissues, making extrapolation of data from other sources difficult. We report a method for producing mast cell-containing single cell suspensions from human intestinal tissue by mechanical and enzymatic dispersion. This method yields 4.5 X 10(5) mast cells per gram of tissue in purity of 3.1 +/- 2.1%. These mast cells were functionally intact as assessed by survival in short-term culture, low spontaneous release, and appropriate IgE-mediated histamine release. They were morphologically intact on electron microscopy and conformed to published descriptions of human lung mast cells. The intestinal mucosal mast cells were also indistinguishable from human lung mast cells in histamine content, goat anti-human IgE dose-response curves, kinetics of histamine release, unresponsiveness to f-met peptide, and production of arachidonic acid metabolites, prostaglandin D2, and leukotriene C4. This procedure produces human intestinal mast cell suspensions in sufficient numbers to make pharmacologic characterization and further purification of this cell feasible.