We examined the regulation of an amiloride-sensitive sodium conductance expressed in human B lymphoid cells. This conductance was activated by two independent pathways, one involving cyclic adenylyl monophosphate (cAMP)- dependent protein kinase and the other involving a pertussis toxin-sensitive G-protein. Cholera toxin, presumably by increasing cellular cAMP, and pertussis toxin, which ADP-ribosylates certain GTP-binding proteins, both independently increased the amiloride-sensitive sodium conductance. Simultaneous treatment with both toxins, however, failed to increase the sodium conductance, implying that a single set of sodium channels was being affected by both toxins. In cells preactivated with pertussis toxin, 8- chlorophenylthio-cAMP inhibited the activated sodium conductance back to the basal level. Thus, cyclic AMP-dependent pathways can either activate or inhibit amiloride-sensitive sodium channels, depending upon the activation state of a pertussis toxin-sensitive GTP-binding protein. These findings support a hypothesis for the regulation of amiloride-sensitive sodium channels which incorporates the independent effects of cholera and pertussis toxins, and in which cyclic AMP can play a dual role in the regulation of channel activity.