Regulation of cytoplasmic free Ca2+ in myocytes is critical to the cardiac cycle Based on the hypothesis that in cardiac myocytes the import into the sarcoplasmic reticulum (SR) of multivalent anions is required for physiological levels of Ca2+ sequestration, we tested the permeability of rat cardiac SR to multivalent anions using light-scattering to monitor osmotically induced changes in microsomal size. The microsomes were permeable to Pi, Fru-6-P, Glc-l-P and Glc-6-P, but not to Man-6-P nor ATP. Disulfonic stilbenes suppressed the permeation to GIc-6-P. The ratiometric Ca2+-sensitive dye Indo-1 was then used to spectrofluorometrically monitor the effects of Pi and G)c-6-P on ATP-dependent Ca2+ sequestration by the microsomes. The sequestration of Ca2+ in the presence of 1 mM ATP was 272±58 pmol/mg protein, while in the presence of 5 mM Glc-6-P + 1 mM ATP and 5 mM Pi + 1 mM ATP were 501±51 and 534±68 pmol/mg protein, respectively. In conclusion, the selective permeability of cardiac microsomes to multivalent anions results in a significant increase in ATP-dependent Ca2+ sequestration Our data suggest that local concentrations of anions such as Glc-6-P may contribute to the regulation of cytoplasmic free Ca2+ in myocytes A contribution from the Fifty 50 Foods Research Program of the Juvenile Diabetes Foundation Int'l.