A set of proteins induced in Pseudomonas aeruginosa PAO1 during growth in the absence of sulfate was characterized by differential two-dimensional electrophoresis and MS. Thirteen proteins were found to be induced de novo or upregulated in P. aeruginosa grown in a succinate/salts medium with sodium cyclohexylsulfamate as the sole sulfur source. Protein spots excised from the two-dimensional gels were analysed by N-terminal Edman sequencing and MS sequencing (MS/MS) of internal protein fragments. The coding sequences for 11 of these proteins were unambiguously identified in the P. aeruginosa genome sequence. Expression of these genes was investigated by reverse transcription-PCR, which confirmed that repression in the presence of sulfate was acting at a transcriptional level. Three classes of sulfur-regulated proteins were found. The first class (five proteins) were high-affinity periplasmic solute-binding proteins with apparent specificity for sulfate and sulfonates. A second class included enzymes involved in sulfonate and sulfate ester metabolism (three proteins). The remaining three proteins appeared to be part of a more general stress response, and included two antioxidant proteins and a putative lipoprotein. This study demonstrates the power of the proteomics approach for direct correlation of the responses of an organism to an environmental stimulus with the genetic structures responsible for that response, and the application of reverse transcription-PCR significantly increases the conclusions that can be drawn from the proteomic study.