Changes in intracellular Ca 2+ concentration ([Ca 2+] i) following the activation of muscarinic receptors with carbachol were studied in cells from the exocrine avian nasal gland that had been maintained in culture for 40–48 h. In these cells, the carbachol-induced sustained increase in [Ca 2+] i could be further increased by the subsequent addition of thapsigargin. This increase was due to an additional release of intracellular Ca 2+ and a corresponding further enhancement of Ca 2+ entry. However, thapsigargin-sensitive and Ins(1,4,5)P 3-sensitive stores appeared to be coincident and the initial carbachol stimulus was sufficient to completely empty these stores. It was concluded that the subsequent effect of thapsigargin was due to a partial refilling of the Ins(1,4,5)P 3-sensitive stores despite the continued presence of agonist, an effect that was not the result of any decline in levels of cellular Ins(1,4,5)P 3 or changes in the generation of Ins(1,3,4,5)P 4, which were sustained throughout. Possible explanations for this refilling response include compartmentalization of intracellular Ins(1,4,5)P 3, or a desensitization of the Ins(1,4,5)P 3 receptor/Ca 2+-release channel. Alternatively, the data are also compatible with a recently proposed kinetic separation of Ca 2+ uptake and release sites. An important implication of this particular interpretation of our findings would be an apparent dependence of Ca 2+ entry specifically on the status of the Ca 2+- uptake component of the agonist-sensitive store, rather than the Ca 2+-release component.