A sensitive and specific GTP-activated Ca2+ translocation process induces rapid Ca2+ movements within cells and appears to reflect G protein-induced membrane fusion or junctional communication between discrete subpopulations of Ca(2+)-pumping organelles (Ghosh, T. K., Mullaney, J. M., Tarazi, F. I., and Gill, D. L. (1989) Nature 340, 236-239). Since fatty acylation can modify G protein action, modification of GTP-induced Ca2+ translocation by fatty acyl-CoA was investigated to throw light on the mechanism underlying Ca2+ transfer. Using permeabilized DDT1MF-2 smooth muscle cells, 2 microM palmitoyl-CoA completely blocked Ca2+ release activated by 20 microM GTP, while having no effect on inositol 1,4,5-trisphosphate-induced Ca2+ release. The IC50 (50% inhibitory concentration) for palmitoyl-CoA was 0.5 microM. Above 3 microM, palmitoyl-CoA inhibited Ca2+ accumulation. Fatty acyl chain length was important, C-13 to C-16 fatty acyl-CoA esters all fully blocking the action of GTP; the IC50 for myristoyl-CoA was also 0.5 microM. C-18 or larger acyl groups had diminished effectiveness as did C-8 or smaller acyl groups. Acetyl-CoA had no blocking effect. In contrast, 10 microM CoA itself blocked GTP-induced Ca2+ release. CoA required a free sulfhydryl group to block, desulfo-CoA having no effect. Removal of ATP by hexokinase and glucose prevented the action of CoA but not palmitoyl-CoA. The free sulfhydryl and ATP requirements indicated CoA was being acylated by endogenous fatty-acyl-CoA synthetase to be effective. The nonhydrolyzable myristoyl-CoA analog, S-(2-oxopentadecyl)-CoA, blocked the GTP effect identically to myristoyl- and palmitoyl-CoA (IC50 = 0.5 microM); thus, fatty acyl transfer is not required, indicating that blockade is due to a direct allosteric modification of a component of the GTP-activated process by acyl-CoA esters. Palmitoyl-CoA not only inhibited but completely reversed GTP-activated Ca2+ release, resulting in the released Ca2+ being taken back up into pools. In the presence of oxalate, GTP-activated Ca2+ transfer results in a substantial increase in Ca2+ accumulation; palmitoyl-CoA also completely reversed this effect resulting in rapid termination of Ca2+ uptake. This reversal provides strong evidence that GTP-activated Ca2+ translocation does not reflect a membrane fusion event. Instead, it likely represents formation of a reversible junction or pore between organelles which may be a required prefusion event.