BACKGROUND/AIMS: Helicobacter pylori (H. pylori) may cause gastric epithelial damage due to secretion of pathogenic proteins. This study was performed to identify proteins secreted by H. pylori and to investigate their cytotoxicity. METHODS: The radioactivity of proteins in the supernatant was compared with that of the intact organism by 2D gel phosphorimaging following 4 hr pulse-chase. The ratio of the amount of UreB, a known cytoplasmic protein, in the supernatant to that in the pellet was found to be 0.22, and this was taken as an index of lysis during the experiment. Values greater than this ratio distinguish proteins that are secreted into the medium, and these proteins were identified by mass spectrometry. After constructing recombinant proteins for these proteins except VacA, the effect of recombinant proteins on the AGS cells were evaluated in the aspect of apoptosis and AGS cell vacuolation using acridine orange uptake in confocal microscopy. RESULTS: Five secreted proteins such as VacA, HP0305, ss-DNA binding 12RNP2 precursor, thioredoxin, HP0902 were identified. The recombinant protein of HP0305 (5microgram/mL) significantly increased apoptosis of AGS cells and AGS cell vacuolation. CONCLUSIONS: Five secreted proteins including VacA were found through the pulse-chase labeling method. The data suggest that HP0305 might have a cytotoxic effect on AGS cells, so play a role in H. pylori pathogenesis in the gastric mucosa.