The non-mitochondrial Ca 2 + stores in permeabilized A7r5 cells responded to a decrease in Mg-ATP concentration with a pronounced Ca 2 + release if 20 μM CoA was present. This release was rather specific for the preincubation or removal of ATP. ATPγS was much less effective and AMP-PNP, GTP, ITP, CTP, UTP, ADP, AMP, adenosine and adenine had no effect. CoA activated with an EC 5 0 of 6 μM. Dephospho-CoA was a less effective cofactor and desulfo-CoA was ineffective. The release induced by Mg-ATP removal did not occur in the presence of 2% fatty acid-free bovine serum albumin and did not develop at 4 o C. All these findings suggest that CoA had to be acylated by endogenous fatty-acyl-CoA synthetase to become effective. Myristoyl- and palmitoyl-CoA esters were identified as the most effective cofactors for the release. Ca 2 + release induced by removing Mg-ATP did not occur if the osmolality of the medium was kept constant by addition of mannitol, sucrose, KCI, MgCl 2 or Mg-GTP, indicating that the decrease in tonicity was the trigger for the release. Mg-ATP plus CoA also synergized with Ca 2 + release induced by a hypotonic shock imposed by diluting the medium with H 2 O. Osmolality changes induced by decreasing the Mg-ATP concentration were more effective in releasing Ca 2 + than equal decreases in concentration of all solutes. We conclude that fatty acyl-CoA esters sensitize the hypotonically induced Ca 2 + release from the non-mitochondrial Ca 2 + stores.