Exposure of Chinese hamster ovary, mouse adrenal cortex tumor (Y-1), THP-1 and U-937 cells and human erythrocytes to adenylate-cyclase-containing urea extracts of Bordetella pertussis (strain 114) organisms promotes the formation of large concentrations of intracellular cAMP. Accumulation is dependent on dose and temperature, with significant accumulation occurring at 4 degrees C, and is virtually instantaneous, with a doubling at 1 min. There is an absolute Ca2+ requirement but external calmodulin (the activator of cyclase activity) has no effect except in erythrocytes and U-937 cells, where it reduces cAMP accumulation. However, calmodulin antagonists inhibit cAMP accumulation. In Y-1 adrenal cells the urea-extract adenylate cyclase stimulates steroidogenesis. Anti-(B. pertussis) antibodies inhibit cyclase activity and prevent further cAMP accumulation after 10 min in cells previously exposed to urea extract. The same effect is obtained by washing. This suggests that a portion of the cyclase is associated with cells in a form not accessible to antibody or washing but accessible to substrate, which we interpret as internalized enzyme with a short lifetime. Continuing cAMP accumulation thus appears to need a continuing source of external cyclase. Inhibitors of the effect of diphtheria toxin, such as NH4Cl, methylamine, chloroquine or monensin, have no inhibitory effect on the accumulation of intracellular cAMP promoted by the internalized adenylate cyclase of urea extracts of B. pertussis organisms. We conclude that entry of the cyclase into cells is not by receptor-mediated endocytosis.