Rat pancreatic zymogen granules were analyzed using flow cytometry to determine their heterogeneity with respect to different characteristics such as size (FSC), internal complexity (SSC) and membrane permeability to lipophilic and cationic dyes using rhodamine-123 as probe. Differences in the chemical composition of the membrane were determined using FITC-labeled lectins (concanavalin A, Wheat germ agglutinin (WGA) and Tetragonolobus purpureus) displaying specific binding for different carbohydrates (D-mannose, N-acetyl D-glucosamine and L-fucose, respectively). Finally, the amylase content of zymogen granules was also analyzed using an anti-amylase antiserum. Our results show the existence of two populations of zymogen granules that can be identified on the basis of their FSC and SSC characteristics: a minor population of approximately 5% all zymogen granules with larger size (FSC) and internal complexity (SSC), and a major population clearly differentiable on the basis of a lower FSC and SSC. Rhodamine-123 uptake was similar in both subpopulations of zymogen granules. By contrast, labeling with fluoresceinated lectins and anti-amylase antiserum showed the existence of a higher content on both amylase and the monosaccharide residues analyzed for the Z2 zymogen granules. However, it is shown that those differences are strongly dependent on the FSC (size) of the granules suggesting that although the carbohydrate contents (D-mannose, N-acetyl D-glucosamine and L-fucose) and the amount of amylase differed from one granule to another, their concentration per granule was similar.