Abstract The fluorescent chelate probe technique is employed in exploring the kinetics of Ca ++ and Sr ++ release from sarcoplasmic reticulum induced by two divalent cation ionophores, A23187 and X537A. Both reagents are shown to release Ca ++ that has been previously accumulated. A23187 is 60X more effective than X537A, while X537A is 100X more effective than A23187 in sequestering Ca ++ into an organic phase in two phase systems. An explanation for the discrepancy between the effectiveness of the antibiotics in the two phase system and the transport rates in sarcoplasmic reticulum is provided by the observation of a particularly low affinity of X537A for Ca ++ and Sr ++ in polar media. The fluorescence properties of A23187 in the vesicle membrane are described.