In this study the effects of changes in extracellular magnesium ([Mg2+]o) and calcium ([Ca2+]o) concentrations on basal and on nerve-mediated and acetylcholine (ACh)-evoked in vitro amylase release and calcium mobilization were investigated in rat parotid gland tissue. In the presence of a normal (2.56 mM) [Ca2+]o, both zero (0 mM) and an elevated (10 mM) [Mg2+]o significantly attenuated basal and ACh-evoked amylase release compared to the response obtained in normal (1.1 mM) [Mg2+]o. During electrical field stimulation (EFS) of parotid tissues, only elevated [Mg2+]o reduced amylase release. In a Ca2+-free medium, both basal and ACh-evoked amylase output were markedly reduced compared to the responses obtained under similar conditions in normal [Ca2+]o. Again, the ACh-induced amylase release in a Ca2+-free solution was larger in normal [Mg2+]o than when the [Mg2+]o was either zero or was elevated to 10 mM. Perturbation of [Mg2+]o had no significant effect on basal intracellular free calcium concentration ([Ca2+]i) in parotid acinar cells loaded with the fluorescent Ca2+ indicator fura-2. Both zero Mg2+ and an elevated [Mg2+]o significantly reduced the ACh-induced rise in the peak and the plateau phase of the Ca2+ transient that was seen in normal [Mg2+]o. In parotid acinar cells loaded with the fluorescent Mg2+ indicator magfura-2, ACh elicited a gradual decrease in intracellular free Mg2+ concentration ([Mg2+]i) to below the basal level. The results indicate that both hypo- and hypermagnesaemia may reduce both basal and ACh-evoked amylase secretion from the salivary gland. As far as the ACh-evoked response is concerned, the effect may be exerted by a decrease in cellular Ca2+ transport. Experimental Physiology (2002) 87.3, 321-326.