The role of Ca 2؉ in stimulus-response coupling in nonexcitable cells is still not well understood. The Ca 2؉ responses of individual cells are extremely diverse, often displaying marked oscillations, and almost nothing is known about the specific features of these Ca 2؉ signals that are important for the functional response of a cell. Using the RBL-2H3 mucosal mast cell as a model, we have studied the temporal relationship between changes in intracellular Ca 2؉ and serotonin secretion at the single-cell level using simultaneous indo-1 photometry and constant potential amperometry. Secretion in response to antigen never occurs until intracellular Ca 2؉ is elevated, nor is it seen during the first few oscillations in Ca 2؉ . Exocytotic events tend to be clustered around the peaks of oscillations, but excellent secretion is also seen in cells with sustained elevations in Ca 2؉ . Ca 2؉ release from stores in the absence of influx fails to elicit secretion. If refilling and continued release of Ca 2؉ from stores is prevented with thapsigargin, Ca 2؉ influx can still trigger secretion, suggesting that store-associated microdomains of Ca 2؉ are not required for exocytosis. Our findings demonstrate the importance of an amplitude-encoded Ca 2؉ signal and Ca 2؉ influx for stimulus-secretion coupling in these nonexcitable cells.Although it is generally agreed that an increase in Ca 2ϩ is both necessary and sufficient for the initiation of secretion in most excitable cells, the role of Ca 2ϩ in nonexcitable cells is less clear (1). The individual Ca 2ϩ responses of nonexcitable cells are often extremely heterogeneous, and many models have been proposed for the generation of these complex and often oscillatory patterns (2). Both amplitude-encoded and frequency-encoded Ca 2ϩ signals have been proposed (3, 4), and the availability of both mechanisms would allow multiple signaling pathways to be activated by Ca 2ϩ in a single cell (5). Hepatocytes, with their repetitive oscillations of constant amplitude but variable frequency are prime candidates for frequencymodulated Ca 2ϩ signaling (6), and it has now been shown that mitochondrial NAD(P)H production in these cells is indeed regulated by the frequency of Ca 2ϩ oscillations (7,8). In contrast, it seems clear that the secretory response of single rat salivary acinar cells is tightly coupled to the amplitude of the Ca 2ϩ response (9), as is ciliary beating in tracheal epithelial cells (10). Furthermore, it has recently been shown that differential activation of transcription factors in B lymphocytes is achieved via non-oscillatory Ca 2ϩ signals of different amplitudes and durations (11).In most cases, however, it has not been easy to determine the specific features of the Ca 2ϩ signal that are important for a physiological response. This is because sensitive methods for detecting function at the single-cell level and with high temporal resolution are not readily available. Furthermore, it now seems clear that additional signals, such as the activation of protein kinase C (12) ...