Glycogen particle synthase phosphatase activity is stimulated by glucose with an A0.5 of approximately 27 mm. The A0.5 is higher than the usual concentrations present in the liver. However, in vitro, certain methylxanthines such as caffeine or theophylline reduce the glucose A0.5 to approximately 10 mm, a concentration well within the normal range of liver glucose concentrations. Methylxanthines do not affect the maximum stimulation by glucose (2.3-fold greater than control rate). The phosphatase reaction also is inhibited by ATP-Mg (I0.5 = 0.1 mM). In the present studies, we have determined the interaction of these effectors. The presence of ATP-Mg at a concentration of 3 mm only slightly reduced the maximal stimulation by glucose. The A0.5 for glucose was unaffected (24 mm). The synergistic effect of caffeine with glucose also was not changed by the presence of ATP-Mg. The A0.5 for glucose was reduced to 11 mm, similar to that in the absence of ATP-Mg. In addition, maximum stimulation by glucose was unchanged. Similar results were obtained when theophylline replaced caffeine. We conclude that the ATP-Mg binding site on either the phosphatase or its substrate, synthase D, does not influence the glucose and methylxanthine binding sites. Effectively, ATP-Mg increased the range over which glucose stimulates the phosphatase activity. In the presence of ATP-Mg, the maximum stimulation by glucose is approximately 7-fold; whereas, in the absence of ATP-Mg it is approximately 2.3-fold. Thus, ATP-Mg may serve to increase the sensitivity of the synthase phosphatase reaction to glucose regulation under in vivo conditions.