The modulation of neuronal adenylylcyclase by Ca2+, acting via calmodulin, is a long-established example of a positive interaction between the Ca2(+)-mobilizing and cAMP-generating systems. In the present study, concentrations of Ca2+ that stimulate brain adenylylcyclase inhibit the adenylylcyclase of NCB-20 plasma membranes. These inhibitory effects of Ca2+ have been characterized and seem to be exerted at the catalytic unit of the enzyme; they are independent of calmodulin, Gi, and phosphodiesterase. To determine whether this inhibition of adenylylcyclase by Ca2+ could occur in the intact cell, cAMP accumulation was measured in response to bradykinin. Bradykinin, which mobilizes Ca2+ in NCB-20 cells, as a consequence of stimulating inositol phosphate production, causes a transient inhibition of prostaglandin E1 stimulation of cAMP accumulation. The inhibitory action of bradykinin is attenuated significantly by treatment of cells with the cell-permeant Ca2+ chelator, 1,2-bis-(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid. It seems likely that the inhibition of adenylylcyclase by low concentrations of Ca2+ represents a novel means for a negative interaction between Ca2(+)-mobilizing and cAMP-generating systems.