Effects of intracellular Mg2+ in the activation of a muscarinic K+ channel were examined in single atrial cells, using patch-recording techniques. In "cell-attached" patch recordings, acetylcholine (ACh) or adenosine (Ado), present in the pipette, activated a specific population of K+ channels. In "inside-out" patches, openings of the K+ channel by ACh or Ado diminished and did not resume until Mg2+ was added to the perfusate which contained GTP or GTP-gamma S, a non-hydrolyzable GTP analogue. Channel openings caused by GTP faded by removing Mg2+, while GTP-gamma S-induced openings persisted steadily even when both Mg2+ and GTP-gamma S were removed. In contrast to the case of GTP-induced channel openings, the GTP-gamma S-induced openings were not inhibited by the A promoter of pertussis toxin with NAD. From these observations, we concluded: Intracellular Mg2+ is essential for GTP to activate the GTP-binding protein. Deactivation of the N protein may be caused by hydrolysis of GTP to GDP. This process may not require Mg2+. During the activation by GTP analogues, the N protein may be dissociated into its subunits.