Many hormones, neurotransmitters and growth factors evoke in their target cells oscillations in the free internal Ca2+ concentration [( Ca2+]i). In electrically non-excitable cells these fluctuations are due to periodic release of Ca2+ from intracellular reservoirs, stimulated by the internal messenger inositol trisphosphate (InsP3). Most models at present invoke fluctuating levels of InsP3 as a key component in generating the oscillations in [Ca2+]i. InsP3 injected into intact cells evokes irregular and transient oscillatory Ca2+-dependent current responses, but the intracellular InsP3 concentration is not constant in such experiments. Here we monitor changes in [Ca2+]i by measuring Ca2+-activated Cl- current in single internally perfused mouse pancreatic acinar cells and show that acetylcholine (ACh), acting through muscarinic receptors, evokes regular and repetitive current pulses which are mimicked by InsP3 applied through a patch pipette. To exclude the possibility that InsP3 is periodically phosphorylated or degraded, we replaced it by the non-metabolizable InsP3 analogue inositol trisphosphorothioate (InsPS3), which also evokes regular pulses of Ca2+-activated Cl- current. These effects are independent of external Ca2+, but abolished by high intracellular concentrations of a Ca2+-chelator. We conclude that repetitive pulses of intracellular Ca2+ release occur even when the concentration of InsP3 is constant.