Tyrosine phosphatase TpbA in Pseudomonas aeruginosa PA14 is a negative regulator of the diguanylate cyclase TpbB. Inactivation of TpbA caused rugose colony morphology which is related to cell persistence in clinical infections. We show here that TpbA is a dual specific tyrosine phosphatase, that TpbB is phosphorylated, and that TpbA controls phosphorylation of TpbB at both Tyr and Ser/Thr residues in vivo as detected by Western blot analysis. In addition, TpbB is demonstrated to be a substrate of TpbA in vitro using purified enzymes. Thus, TpbA controls the rugose morphology in P. aeruginosa by dephosphorylating TpbB.