A central unresolved question in agonist-evoked [Ca2+]i signaling is the pathway by which [Ca2+]i oscillations and a sustained response are transduced. We show here that activation of Gβγ signal [Ca2+]i oscillations and activation of Gαq signal a sustained response during stimulation by a number of Ca2+-mobilizing agonists. Thus, infusion of purified Gβγ into pancreatic acinar cells through a patch pipette evokes [Ca2+]i oscillations by Ca2+ release from internal stores, which were inhibited by two independent scavengers of Gβγ, the β-adrenergic receptor kinase fragment, and a mutated Gαi1G203A. These proteins, as well as an inhibitory antibody against Gαq/11, prevent [Ca2+]i oscillations and the sustained response when applied before cell stimulation, possibly by preventing the dissociation of Gq into its subunits. After cell stimulation and dissociation of Gq into Gβγ and Gαq, scavenging Gβγ stabilized the sustained response and inhibited reassociation of the subunits on termination of cell stimulation with antagonist, whereas scavenging Gαq inhibited the sustained response and uncovered the Gβγ-dependent oscillations. These findings provide a general mechanism by which Ca2+-mobilizing agonists can control the type of [Ca2+]i signal to be transduced to the cell interior.