Activation of particulate adenylate cyclase and detergent-soluble (lubrol PX) adenylate cyclase occurred when the enzyme was preincubated at 37 degrees in the presence of 5 mM NaF and 5mM MgSO4. Under these conditions the specific activity of the enzyme increased more than twofold in 8 to 12 min. Activation also occurred in the presence of 5 mM NaF alone, but the rate of activation was slower. Under these conditions, activation was inhibited by 1mM EDTA, but this inhibition was prevented byMg++. No activation was observed at 0 degrees or in the absence of F. After repeated extraction with detergent, particulate adenylate cyclase was not stimulated by mM NaF, and activation by preincubation with Mg++ and F- was significantly reduced. Activation was restored by recombination of this particulate fraction with the initial detergent extract. This activating effect appeared to be mediated by one or more proteins present in the detergent extract.