Ion-exchange chromatography of rabbit heart adenylate cyclase solubilized with lubrol PX results in two peaks of activity, AC I and AC II, differing in their sensitivity to guanylylimido-diphosphate [Gpp(NH)p], NaF and cholera toxin. AC I is activated 4- to 5-fold by Gpp(NH)p, 10- to 20-fold by NaF and 3- to 4-fold by cholera toxin. AC II is insensitive to Gpp(NH)p and cholera toxin, but is activated 2-fold by the fluoride. The differences in the regulatory properties of AC I and AC II are probably due to the unequal distribution of the N-protein, the regulatory component of adenylate cyclase, in the preparations. Addition of the N-protein, obtained by thermoinactivation of AC I, to AC II results in restoration of the enzyme sensitivity to Gpp(NH)p as well as in an increase of the stimulating effect of NaF. It is shown that the lag-period observed during adenylate cyclase stimulation by Gpp(NH)p is due to the slow transition of the N-protein into an activated conformation. This Gpp(NH)p-activated N-protein interacts with the catalytic component of adenylate cyclase without any lag-period. Addition of GTP to the Gpp(NH)p-activated soluble adenylate cyclase complex leads to a decrease of the enzymatic activity. This process presumably occurs via substitution of Gpp(NH)p for GTP in the N-protein with subsequent hydrolysis of GTP and not by exchange of the N-protein-Gpp(NH)p complex for the N-protein-GTP complex within the catalytic component of adenylate cyclase.