The effects of ACh on [3H]D-aspartate efflux and on calcium levels ([Ca2+]i) were studied at the same time in sister cultures of rat cerebellar granule cells stimulated with electrical pulses (5-20 Hz) or depolarized with KCl (15-40 mM). ACh, 0.3-1000 nM, greatly facilitated the 10-Hz-evoked tritium efflux while its effect on 20 mM KCl-evoked efflux was significantly smaller. ACh, 10-1000 nM, enhanced [Ca2+]i levels to a limited extent under both experimental conditions. Therefore, ACh facilitation was evident above all on the electrically evoked [3H]D-aspartate efflux. The ACh-mediated responses depended on the activation of M3-muscarinic receptors since these responses were blocked by 4-DAMP. ACh, 50 microM, reduced the [Ca2+]i plateau, determined by prolonged electrical or KCl stimulation. This effect was due to its action of M2-receptors being blocked by AF-DX 116. In conclusion, at very low concentrations, ACh greatly facilitated the electrically evoked [3H]D-aspartate efflux through M3-receptors, while at a higher concentrations, it inhibited, through M2-receptors, the rise in [Ca2+]i caused by prolonged cell depolarization.