We have analyzed the proteome of several strains of Haemophilus ducreyi by two-dimensional gel electrophoresis (2-DE) and mass spectrometry. Over 100 spots were analyzed from the soluble and insoluble protein fractions from the prototype strain 35000HP and 122 distinct proteins were identified. Functions of approximately 80% of the 122 proteins were deduced by identification with close homologues of Haemophilus influenzae. Four additional wild type and three mutant strains were also analyzed that vary in their virulence and/or outer-membrane lipooligosaccharide structures. Overall, the 2-DE gel maps of the wild type and mutant strains were similar to strain 35000HP, suggesting little proteome diversity in relation to carbohydrate expression and/or virulence. An exception was the Kenyan strain 33921 which contained significant differences in its proteome 2-DE map and also synthesizes an unusual LOS with a trisaccharide branch structure. This African strain may represent a prototype of a second clonal group of H. ducreyi.