The newly recognized bacterial second messenger 3′,5′-cyclic diguanylic acid (cyclic diguanylate (c-di-GMP)) has been shown to regulate a wide variety of bacterial behaviors and traits. Biosynthesis and degradation of c-di-GMP have been attributed to the GGDEF and EAL protein domains, respectively, based primarily on genetic evidence. Whereas the GGDEF domain was demonstrated to possess diguanylate cyclase activity in vitro, the EAL domain has not been tested directly for c-di-GMP phosphodiesterase activity. This study describes the analysis of c-di-GMP hydrolysis by an EAL domain protein in a purified system. The Vibrio cholerae EAL domain protein VieA has been shown to inversely regulate biofilm-specific genes (vps) and virulence genes (ctxA), presumably by decreasing the cellular pool of c-di-GMP. VieA was maximally active at neutral pH, physiological ionic strength, and ambient temperatures and demonstrated c-di-GMP hydrolytic activity with a K m of 0.06 μM. VieA was unable to hydrolyze cGMP. The putative metal coordination site of the EAL domain, Glu 170, was demonstrated to be necessary for VieA activity. Furthermore, the divalent cations Mg 2+ and Mn 2+ were necessary for VieA activity; conversely, Ca 2+ and Zn 2+ were potent inhibitors of the VieA phosphodiesterase. Calcium inhibition of the VieA EAL domain provides a potential mechanism for regulation of c-di-GMP degradation.