A panel of rat monoclonal antibodies was used to classify mouse mast cells from four different sources on the basis of their surface determinants. Three populations of mast cells were derived by culturing normal bone marrow, spleen, and peripheral blood cells, whereas the fourth population was isolated directly from the peritoneal cavity. For each population, a surface antigen phenotype was determined by indirect immunofluorescence microscopy and compared to the phenotypes of the other populations of mast cells. The results showed that culture-derived mast cells, regardless of origin, had nearly identical antigen phenotypes, suggesting that these populations were comparable. By contrast, peritoneal mast cells had a different phenotype, suggesting that they represented a class distinct from culture-derived mast cells. These findings, which corroborate classification schemes based on nonimmunologic methods, demonstrate that subpopulations of mouse mast cells can be distinguished antigenically.