The role of cAMP in the regulation of the high affinity choline uptake (HACU) was investigated in resting and KCl-stimulated rat brain synaptosomes. The data indicate that the permeable cAMP analogue, monobutyryl-8-bromo cAMP, increased dose-dependently the HACU in resting synaptosomes. Treatments of resting synaptosomes by oxotremorine, quinacrine, and promethazine resulted in a reduced cAMP formation with a concomitant decrease of HACU. The reduction of HACU could be completely counteracted by the monobutyryl-8-bromo cAMP following oxotremorine treatment and was only partially inhibited in quinacrine and promethazine treated resting synaptosomes. KCl stimulation resulted in a significant increase in cAMP formation and HACU by the synaptosomes. The different profile of data obtained following the previous pharmacological treatments in KCl-stimulated synaptosomes suggests that both cAMP and phospholipase A2 pathways may act synergistically to coordinate the neuronal choline incorporation.