A well-differentiated dog mastocytoma was characterized ultrastructurally using morphometric, histochemical, and biochemical methods. The ultrastructure of cells in the intact tumor was compared to the morphology of cells disaggregated from the tumor and cultured for periods as long as 4 weeks and to normal dog connective tissue mast cells. Most of the tumor cells contained histamine (mean = 5.81 pg/cell), demonstrated chloroacetate esterase activity histochemically, stained metachromatically with toluidine blue, and were similar in ultrastructure to normal dog mast cells. The proportion of mast cells in this tumor averaged 67%; eosinophils, fibroblasts, plasma cells, and macrophages also were present. The mean diameter of mast cells (12.79 micron) and the mean diameter of their cytoplasmic granules (473 nm) were similar to those reported for mast cells and mastocytoma cells from various species. The heterogeneity in appearance of the mastocytoma granules is consistent with a variable degree of granule maturation. After disaggregation or periods of culture ranging from 2 days to 4 weeks, the mean granule diameters were 15% larger than those measured in the intact mastocytoma cells, though other morphological features remained unchanged. Although the cells retained their distinct morphological features for at least 4 weeks, some of their physiological responses were lost after 1 week in culture. Our study showed that dog mastocytomas can be a source of a large, relatively homogeneous population of cells that are useful for elucidating some of the structural and functional properties of mast cells.