Helicobacter pylori is an important human gastrointestinal pathogenic bacterium which is believed to colonize approximately one-half of the world's population. Different strains of H. pylori possess virulence proteins for tissue colonization, host evasion and tissue damage. The bacteria display genomic instabilities that include gene rearrangements and gene exchange. Recently, methods for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) have been established for monitoring biomarkers in bacterial extracts. In order to establish a set of H. pylori specific biomarkers as well as a set of strain-specific biomarkers, we examined lysates and extracts from six different strains of this bacterium. Three different MALDI matrices, a-cyano-4-hydroxycinnamic acid, sinapinic acid, and ferulic acid were tested for sensitivity of analysis. Also, the effects of solubilizing analytes with the detergent noctyl-b-D-glucopyranoside were explored. It was found that a set of H. pylori specific, and a probable set of strain-specific, biomarkers could be established using MALDI-TOFMS. The use of H. pylori fingerprinting by MALDI may be useful for typing of these bacteria, or for studying genetic drift at the phenotypic level in specific strains.