In a previous report it was shown that EDTA inhibition of liver glycogen synthase phosphatase activity in preparations from normal, fed rats could be increased upon glucagon or cAMP treatment. This occurred without a change in the half-maximum inhibitory concentration of EDTA. Glucose administration to animals resulted in decreased EDTA inhibition. The inhibitory action of EDTA has been further characterized by comparing its action with that of other chelators (CDTA and EGTA) and examining the effects of various divalent cations on chelator inhibition. Both CDTA and EDTA which differ structurally were inhibitory at 5 m m concentrations whereas EGTA which is structurally similar to EDTA was not inhibitory at concentrations up to 10 m m. The lack of inhibition by EGTA could be explained by its weak affinity for Mg++ in the preparation. A comparison of CDTA and EDTA revealed that CDTA was a more potent inhibitor than EDTA (I0.5, 0.15 m m vs 0.3 m m). Glucagon and glucose treatment of rats resulted in changes in CDTA inhibition which closely paralleled those of EDTA. A large group of divalent cations were tested but only Mg++, Ca++, and Mn++ both prevented and reversed CDTA or EDTA inhibition. Fifty percent reversal using either chelator occurred at calculated free-metal ion concentrations of approximately 2 μm, 0.08 μm and 0.0004 μm, respectively. Thus, it is clear that EDTA inhibition is due to its chelation effect and is not due to a nonspecific anionic effect.