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Helicobacter pylori extracellular proteins are of interest because of possible roles in pathogenesis, host recognition, and vaccine development. We utilized a unique approach by growing two strains (including one nonsequenced strain) in a defined serum-free medium and directly analyzing the proteins present in the culture supernatants by LC-MS/MS. Over 125 proteins were identified in the extracellular proteomes of two H. pylori strains. Forty-five of these proteins were enriched in the extracellular fraction when compared to soluble cell-associated protein samples. Our analysis confirmed and expanded on the previously reported H. pylori extracellular proteome. Extracellular proteins of interest identified here included cag pathogenicity island protein Cag24 (CagD); proteases HP0657 and HP1012; a polysaccharide deacetylase, HP0310, possibly involved in the hydrolysis of acetyl groups from host N-acetylglucosamine residues or from residues on the cell surface; and HP0953, an uncharacterized protein that appears to be restricted to Helicobacter species that colonize the gastric mucosa. In addition, our analysis found eight previously unidentified outer membrane proteins and two lipoproteins that could be important cell surface proteins.