The cAMP receptor protein (CRP) positively regulates the expression of Vibrio vulnificus putAP genes encoding a proline dehydrogenase and a proline permease. In the present study, an open reading frame encoding PutR was identified downstream of the putAP genes and a mutational analysis revealed that the PutR protein was also involved in regulating the putAP transcription by activating Pput promoter. Although CRP acts as a primary activator and the influence of PutR on Pput is mediated by CRP, the level of Pput activity observed when PutR and CRP functioned together was greater than the sum of Pput activities achieved by each activator alone. Western blot analyses demonstrated that the cellular levels of PutR and CRP were not significantly affected by each other, indicating that PutR and CRP coactivate Pput rather than function sequentially in a regulatory cascade. Two adjacent binding sites for PutR mapped by in vitro DNase I protection assays were found to overlap the CRP binding sites and were centred -91.5 (PCBI) and -133.5 bp (PCBII) upstream of the transcription start site of Pput respectively. PutR and CRP bind to the sites cooperatively and a dissection of the role of the binding sites revealed that CRP at PCBI plays the most crucial role in the activation of Pput. Accordingly, the present results revealed that PutR and CRP coactivate the expression of Pput and exert their effect by cooperatively binding to the promoter.