Abstract: The effect of inositol 1,4,5‐trisphosphate [Ins‐(1,4,5)P₃] and caffeine on Ca²⁺ release from digitonin‐per‐meabilised bovine adrenal chromaffin cells was examined by using the Ca²⁺ indicator fura‐2 to monitor [Ca²⁺]. Permea‐bilised cells accumulated Ca²⁺ in the presence of ATP and addition of either Ins(1,4,5)P₃ or caffeine released 17% or 40–50%, respectively, of the accumulated Ca²⁺, indicated by sustained rises in [Ca²⁺] in the cell suspension. Prior addition of Ins(l,4,5)P₃ had no effect on the magnitude of the response to a subsequent addition of caffeine. The response to Ins(l,4,5)P₃ was prevented by prior addition of caffeine or CaCl₂, indicating that the Ins(l,4,5)P₃ response was blocked by elevated [Ca²⁺]. The responses were essentially identical in the presence of the proton ionophore carbonyl cyanide m‐chlorophenylhydrazone, indicating that the Ca²⁺ release was not from mitochondria or secretory granules and that a proton gradient was not required for Ca²⁺ accumulation into the Ins(l,4,5)P₃‐ or caffeine‐sensitive stores. Ca²⁺ release from the caffeine‐sensitive store was selectively blocked by ryano‐dine. The Ins(l,4,5)P₃‐sensitive store was emptied by thap‐sigargin, which had no effect on caffeine responses. These data suggest that permeabilised chromaffin cells possess two distinct nonoverlapping Ca²⁺ stores sensitive to either Ins(1,4,5)P₃ or caffeine and support previous conclusions that these stores possess different Ca²⁺‐ATPases.