Helicobacter pylori is a major human pathogen that is responsible for a number of gastrointestinal infections. We have used 2-DE to characterise protein synthesis in bacteria grown either on solid agar-based media or in each of two broth culture media (Brucella and brain heart infusion (BHI) broth). Significant differences were observed in the proteomes of bacteria grown either on agar-based or in broth media. Major changes in protein abundance were identified using principal component analysis (PCA), which delineated the profiles derived for the three key growth conditions (i.e. agar plates, Brucella and BHI broth). Proteins detected across the gel series were identified by peptide mass mapping and Edman sequencing. A number of proteins associated with protein synthesis in general as well as specific amino acid synthesis were depressed in broth-grown bacteria compared to plate-grown bacteria. A similar reduction was also observed in the abundance of proteins involved in detoxification. Two of the most abundant spots, identified as UreB and GroEL, in plate-grown bacteria showed a >140-fold drop in abundance in bacteria grown in Brucella broth compared to bacteria grown on agar plates. Two protein spots induced in bacteria grown in broth culture were both identified as glyceraldehyde 3-phosphate dehydrogenase based on their N-terminal amino acid sequences derived by Edman degradation. The underlying causes of the changes in the proteins abundance were not clear, but it was likely that a significant proportion of the changes were due to the alkaline pH of the broth culture media.