In vitro assay of the adenylate cyclase of NB41A neuroblastoma cells in the presence of increasing concentrations of MnClz suggested that the enzyme is modulated by both high-and low-affinity sites for manganese. MnCI2 in a concentration of 1 pM significantly stimulated adenylate cyclase activity, but increasing the concentration of manganese to 3 pM or 10 p. M had no further effect. Raising MnCI2 to 0.1 or 1 mM, however, further stimulated enzyme activity. In addition to differences in affinity for manganese, the two classes of binding sites may be distinguished by differences in their interaction with other agents that affect adenylate cyclase activity. Millimolar manganese and magnesium appeared to compete for a common site on the enzyme and the effect of manganese in this range and the effect of guanyl nucleotide were synergistic. In contrast, the stimulation of activity by micromolar manganese appeared to be additive to the effects of either increasing magnesium or the addition of guanyl nucleotide to the assay media. Comparison of the substrate dependency of the reaction measured in the presence and absence of manganese suggests that the stimulation of adenylate cyclase activity involves increases in both the apparent Vmax of the reaction and the affinity for ATP. The results raise the possibility that the interaction of Mn2+ may play a role in the modulation of adenylate cyclase in vivo.