Vibrio cholerae is the agent of the severe diarrheal disease cholera, and it perpetuates in aquatic reservoirs when not in the host. Within the host's intestines, the bacteria execute a complex regulatory pathway culminating with the production of virulence factors that allow colonization and cause disease. The ability of V. cholerae to form biofilms is thought to aid its persistence in the aquatic environment and passage through the gastric acid barrier of the stomach. The transcriptional activators VpsR and VpsT are part of the biofilm formation-regulatory network. In this study, we screened a V. cholerae genomic library in Escherichia coli cells containing a P(vpsT)-luxCDBAE transcriptional fusion reporter and found that a plasmid clone containing the aphA gene activates the expression of vpsT in E. coli. AphA is a master virulence regulator in V. cholerae that is required to activate the expression of tcpP, whose gene products in turn activate all virulence genes including those responsible for the synthesis of the toxin-coregulated pilus (TCP) and cholera toxin through the activation of toxT. AphA has a direct effect on the vpsT promoter, as gel shift experiments demonstrated that AphA binds to the vpsT promoter region. Furthermore, V. cholerae aphA mutants exhibit significantly lower levels of vpsT expression as well as reduced biofilm formation. AphA thus links the expression of virulence and biofilm synthesis genes.