In this study the effects of changes in extracellular magnesium ([Mg(2+)](o)) and calcium ([Ca(2+)](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) [Ca(2+)](o), both zero (0 mM) and an elevated (10 mM) [Mg(2+)](o) significantly attenuated basal and ACh-evoked amylase release compared to the response obtained in normal (1.1 mM) [Mg(2+)](o). During electrical field stimulation (EFS) of parotid tissues, only elevated [Mg(2+)](o) reduced amylase release. In a Ca(2+)-free medium, both basal and ACh-evoked amylase output were markedly reduced compared to the responses obtained under similar conditions in normal [Ca(2+)](o). Again, the ACh-induced amylase release in a Ca(2+)-free solution was larger in normal [Mg(2+)](o) than when the [Mg(2+)](o) was either zero or was elevated to 10 mM. Perturbation of [Mg(2+)](o) had no significant effect on basal intracellular free calcium concentration ([Ca(2+)](i)) in parotid acinar cells loaded with the fluorescent Ca(2+) indicator fura-2. Both zero Mg(2+) and an elevated [Mg(2+)](o) significantly reduced the ACh-induced rise in the peak and the plateau phase of the Ca(2+) transient that was seen in normal [Mg(2+)](o). In parotid acinar cells loaded with the fluorescent Mg(2+) indicator magfura-2, ACh elicited a gradual decrease in intracellular free Mg(2+) concentration ([Mg(2+)](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 Ca(2+) transport.