EDTA at a concentration of 1 muM produced a threshold effect in the activation of purified rabbit liver fructose-1, 6-diphosphatase [EC 3.1.3.11] in the presence of 5 mM Mg2+ at pH 7.2. Without EDTA, biphasic activation curves were produced by Mg2+. A double-reciprocal plot of the data gave the Km values corresponding to the two linear regions. They were 0.19 and 0.83 mM at pH 7.5, and 0.055 and 0.83 mM at pH 9.1. In the presence of 5muM EDTA a sigmoidal curve was obtained for Mg2+ activation in the range of noninhibitory Mg2+ concentrations at pH 7.2. The apparent Km value for Mg2+ was 0.15 mM, and the Hill coefficient was 2.0. At pH 9.1 cooperativity among the Mg2+ sites disappeared, and the apparent Km value for Mg2+ was 0.055 mM. These Km values at pH 7.2 or 9.1 corresponded to the smaller of the biphasic Km values obtained without EDTA. In the absence of EDTA, no inhibition by Mg2+ was observed in the Mg2+ concentration range below 10 mM. In the presence of EDTA, the enzyme was inhibited markedly by Mg2+ at concentrations above 0.5 mM at pH 7.2, and was more sensitive to inhibition at pH 9.1. The effects of pH on the Km value for Mg2+ activation and on the Mg2+ inhibition contributed to an apparent shift of the pH optimum for activity induced by EDTA. Cooperative interaction among fructose-1, 6-diphosphate sites was observed for the enzyme in the presence of EDTA. The Hill coefficient was approximatley 1.8, and the apparent Km value for the substrate was 0.74 muM. EDTA appears to make liver fructose-1, 6-diphosphatase very sensitive to various effectors. It is suggested that Mg2+ serves as a regulator for the enzyme activity.