In pancreatic acinar cells, as in many other cell types, the tumour promoter thapsigargin (TG) evokes a significant increase of intracellular free Ca2+ ([Ca2+](i)). The increases of [Ca2+](i) evoked by TG was associated with significant changes of plasma membrane Ca2+ permeability, with [Ca2+](i) values following changes in extracellular [Ca2+]. Plasma membrane Ca2+ extrusion is activated rapidly as a consequence of the rise in [Ca2+](i) evoked by TG and the rate of extrusion is linearly dependent on [Ca2+](i) up to 1 mu M Ca2+. In contrast, the activation of the Ca2+ entry pathway is delayed and the apparent rate of Ca2+ entry is independent of [Ca2+](i). In the presence of 20 mM caffeine, which reduces the resting levels of inositol trisphosphate (InsP(3)), the increase of [Ca2+](i) evoked by TG was significantly reduced. The reduction was manifest both as a decrease of the amplitude of the [Ca2+](i) peak (30% reduction) and, more importantly, as a reduction of the apparent maximal rate of [Ca2+](i) increase (from 12.3+/-1.0 to 6.1+/-0.6 nM Ca2+/s). The inhibition evoked by caffeine was reversible and the removal of caffeine in the continuous presence of TG evoked a further increase of [Ca2+](i). The amplitude of the [Ca2+](i) increase upon caffeine removal was reduced as a function of the time of TG exposure. Addition of TG in the presence of 1 mM La3+, which is known to inhibit the plasma membrane Ca2+-activated adenosine triphosphatase, induced a much higher peak of [Ca2+](i). This increase was associated with an augmentation of the apparent rate of [Ca2+](i) increase (from 12.3 +/- 1.2 to 16.1 +/- 1.9 nM Ca2+/s). The inhibitory effect of caffeine, as well as the increase in [Ca2+](i) observed on caffeine removal was not affected by the presence of 1 mM La3+. These data indicate that an important component of the TG-evoked [Ca2+](i) increase is due to InsP(3)-sensitive Ca2+ release which is probably mediated by the resting levels of InsP(3).