AbstractRelease of calcium from intracellular stores of rat basophilic leukemia cells was monitored using the fluorescent probe chlortetracycline. The ability of chlortet‐racycline to indicate release from intracellular calcium stores was initially validated. The decrease of chlortetracycline fluorescence upon antigen‐stimulation was not the result of secretion of granule‐associated dye or of changes in the properties of the membranes. The chlortetracycline fluorescence signal was not influenced by Ca2+influx across the plasma membrane. Results obtained from these chlortetracycline fluorescence measurements corresponded well with45Ca efflux data, an indirect measurement of release of calcium from stores. Chlortet‐racycline was used to examine the rate of antigen‐induced release of calcium from stores, the depletion of intracellular calcium stores by EGTA, and the relationship between the antigen‐stimulated release of stored calcium and exocytosis. Chlortetracycline was shown to be a useful qualitative indicator for the release of intracellular calcium with a relatively rapid re