Acetylcholine (Ach) was tested for its effect on calcium currents in primary cultures of embryonic rat hippocampal neurones. 10 microM Ach reversibly depressed the high voltage activated (HVA) Ca current and the effect was blocked by atropine. When GTP binding proteins (G-proteins) were irreversibly activated by intracellular perfusing the neurons with GTP[S], one short-lasting application of Ach permanently inhibited Ca currents. Alternatively, the transmitter was inactive when the cells were preincubated with pertussis toxin, which inactivates some G-proteins by catalyzing ADP ribosylation. Our results suggest that a G-protein mediates the inhibitory modulation of the HVA Ca current by muscarinic agonists.