The protein expression profile of a novel marine oligotrophic ultramicrobacterium, Sphingomonas sp. strain RB2256, was investigated by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). Analytical reference maps were generated from mid-log phase batches and steady-state chemostat cultures with pH 4-8 immobilised pH gradients (IPGs) followed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The resolved proteins were detected by two different methods: radioactive labeling and silver staining. Protein profiles generated from analytical 2-D PAGE gels were compared and differential analysis was performed using Melanie II software. Both methods (radioactive labeling and silver staining) resulted in reproducible, high resolution gels (up to 1600 protein spots). This approach is proving to be a powerful tool for investigating the molecular basis of the unique physiology of this model oligotrophic microorganism.