1. The effects of acetylcholine (ACh) on granule cells freshly dissociated from rat dentate gyrus (DG) were studied using the nystatin perforated patch technique. This method allowed us to study ACh-induced currents (IACh) under voltage clamp without "run-down" of the ACh response. In some experiments, we used the conventional whole-cell method for intracellular application of drugs not permeable to cell membrane. 2. At a holding potential of -40 mV, ACh induced an outward current. The amplitude of IACh increased in a sigmoidal fashion with increasing ACh concentration. The half-maximal response and the Hill coefficient determined from the relation between ACh concentration and response were 4.98 x 10(-7) M and 1.70, respectively. 3. The reversal potential of IACh was close to the K+ equilibrium potential. The IACh was accompanied by an enhancement of the K+ current. 4. Muscarine and McN-A-343 mimicked the ACh response, whereas oxotremorine induced no response. 5. Muscarinic antagonists reversibly suppressed the IACh (10(-5) M) in a concentration-dependent manner, where the values of half-inhibition concentration (IC50) were 1.03 x 10(-6) M for pirenzepine and 2.21 x 10(-5) M for AF-DX-116. 6. Intracellular perfusion with GDP-beta S suppressed the IACh greatly. The IACh persisted in the neurons pretreated with an external solution containing pertussis toxin (IAP) for 18 h. 7. In the neurons perfused with Ca(2+)-free external solution containing 2 mM ethylene glycol-O,O'-bis (beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid and 10 mM Mg2+, the first application of ACh induced the IACh with an amplitude similar to that in the standard solution.(ABSTRACT TRUNCATED AT 250 WORDS)