: We found numerous small vesicles released from the cell by thin sectioning of the plate culture of Vibrio cholerae and V. parahaemolyticus fixed with the freeze-substitution technique. From the broth media of exponentially growing bacteria we could collect the vesicles by the centrifugation but not enough without fixation. The vesicles are encompassed with a membrane structure similar to the outer membrane of these bacteria. The anti-O (Inaba) serum reacted with the surface of the vesicles and the inside of the vesicle are generally filled with an electron-dense mass.Key words : Outer membrane, Vesicle, Vibrio cholerae, Vibrio parahaemolyticus, Protein secretion Release of membrane-bound vesicles from Gram-negative bacteria has been seen in several bacterial species (2,3,9,10,14). Since the vesicles are usually associated with some of the biological activity such as endotoxin, bacteriocin and enzymatic activities, the relationship of vesicle production and pathogenicity of the bacteria has also discussed (8). The evidence of the association of the vesicles with some biological activities suggests that these active substances might be secreted from the bacterial cells concomitantly with the release of the vesicles.In our morphological observations of the cell structure of Vibrio cholerae and V. parahaemolyticus we found many small vesicles in the culture medium of these bacteria. In this communication we described the morphological aspects of these vesicles released from Vibrio species and discuss their relationship to the protein secretion mechanisms.V. cholerae strain TSI-4 biotype classical and serotype Inaba, and V. parahaemolyticus strain WP-4 were used. These strains were cultured in L broth or on L agar plate at 37 C.For electron microscopy, thin sectioning was carried out by the freeze-substitution technique using liquid nitrogen as a cryogen following the method of Amako et al (1, 12). For the negative staining, the bacteria cultured on an agar plate were fixed with 1% glutaraldehyde and a portion of the bacteria were suspended in a 2% solution of ammonium acetate and negatively stained with 1% potassium phosphotungstate.A thin-sectioned profile of a colony on an L agar plate of strain WP-1 of V. parahaemolyticus and strain TSI-4 of V. cholerae are shown in Figs. 1 and 2, respectively. Many small vesicles were found in the extracellular space between the bacterial cells. The vesicles were rather uniform in size, and the average size of the diameter of the vesicles of V. cholerae was 22 nm. They were encompassed with a unit membrane structure and contained an electron-dense mass (Fig. 2). The size and the shape of the unit membrane were similar to those of the outer membrane. It was possible that the vesicles found in the colony were degradation products released from autolytically disintegrated cells on the agar plate. To eliminate this possibility the culture medium of bacteria in exponentially growing in aerated liquid culture was examined for the presence of vesicles. V. cholerae strain TSI...