The relationship between intracellular free calcium activity ([Ca2+]i) and stimulated amylase secretion was investigated in rat parotid acini by measuring the effects of substance P methyl ester and isoprenaline on quin2 fluorescence and amylase release. Although both of these drugs evoked concentration-dependent increases in [Ca2+]i and amylase release, the tachykinin had a greater effect on [Ca2+]i while the catecholamine was the more effective secretagogue. Whereas isoprenaline exerted equipotent effects on amylase secretion and [Ca2+]i, the dose-response relationship for stimulation of secretion by substance P was dissociated by three orders of magnitude to the right of that for elevation of [Ca2+]i by this peptide. It is concluded that these data do not support the hypothesis that substance P-stimulated amylase secretion is mediated solely through an increase in [Ca2+]i and that other second-messengers may be involved in mediation of this secretory response.