The two component system PhoB/PhoR controls expression of genes of the Pho regulon, and the phoBR operon itself. Under low phosphate levels PhoR is phosphorylated, PhoR~Pi transfers Pi to PhoB (response regulator), which  binds to the Pho box sequences in the promoter regions of many genes. V. cholerae has a functional PhoB/PhoR system essential for Pi uptake and the intestinal colonization. In the present work we analyzed the autoregulation of phoBR operon of V. cholerae O1, and investigated aditional roles of PhoB/PhoR in the bacteria. A search for putative Pho boxes in a 250bp long strech of the putative phoBR promoter with ClustalW showed three putative binding sequences: a typical at -35 in the sense orientation and two at -65 and -175 in the antisense orientation. By similar approach, promoter regions of other genes were inspected. Three genes were chosen as putative members of the V. cholerae Pho regulon: (a) iciA, whose transcription activation is PhoB-dependent and controls DNA replication in E. coli; (b) surE, required for stationary-phase survival of E. coli and (c) aphB, a component of the V. cholerae virulence cascade. Eletrophoretic mobility shift assays (EMSA) of the PhoB binding to 234 bp of the phoBR putative promoter showed three bands of retardation, suggesting three possible binding sites for PhoB. Surprinsingly, promoter-lacZ fusion showed that the sequence of 50 bp containing only the Pho box at -35 activated lacZ transcription more intensively than the 234bp. A lacZ fusion of 170bp of the promoter containing the two putative Pho boxes at -35 and -65 showed extensive repression of of lacZ transcrition, with respect to the -35 box alone, suggesting a repressive role for the -65 sequence. Atomic force microscopy confirmed PhoB binding to the 234 bp region and showed a bend in the DNA when the protein binds to multiple sites. EMSA with the putative promoters of iciA, surE and aphB showed specific binding of PhoB. PhoB binding was confirmed by lacZ fusion assay of the promoters activities of these sequences in a V. cholerae background, under low Pi levels, indicating a transcriptional activation of iciA, surE and aphB by PhoB. The results presented here suggest that PhoB probably has an activation role in important processes in addition to phosphate metabolism.