Serine esterases were detected in the granules of mucosal mast cells from rat, mouse, sheep, and man. Successful demonstration of enzyme activity required brief fixation (6 h) of tissues in 4% paraformaldehyde. Staining with naphthol AS-D chloroacetate produced an intense red reaction product in intraepithelial mucosal mast cells (globule leucocytes) and mucosal mast cells within the lamina propria of the gastrointestinal tract. The mast cell identity of cells stained for esterase was confirmed by sequential staining with toluidine blue (pH 0.5). Furthermore, the numbers of cells detected after staining for esterases or with toluidine blue were highly correlated. Esterase activity within mucosal mast cells/globule leucocytes from all species was inhibited with the serine enzyme inhibitor phenylmethylsulphonyl fluoride. Further histochemical studies with the substrate, N-acetyl-DL-phenylalanine B-naphthyl ester, indicated that mucosal mast cells and globule leucocytes contain esterases which are chymotrypsin like in substrate specificity.