Recent studies indicate that the Ca²⁺ permeability of the sarcoplasmic reticulum (SR) can be affected by its anionic environment. Additionally, anions could directly modulate the SR Ca²⁺ pump or the movement of compensatory charge across the SR membrane during Ca²⁺ uptake or release. To examine the effect of anion substitution on cardiac SR Ca²⁺ uptake, fluorometric Ca²⁺ measurements and spectrophotometric ATPase assays were used. Ca²⁺ uptake into SR vesicles was inhibited in a concentration-dependent manner when Br or I replaced extravesicular Cl (when Br completely replaced Cl, uptake velocity was ~ 70% of control; when I completely replaced Cl, uptake velocity was ~ 39% of control). Replacement of Cl with SO had no effect on SR uptake. Although both I and Br inhibited net Ca²⁺ uptake, neither anion directly inhibited the SR Ca²⁺ pump nor did they increase the permeability of the SR membrane to Ca²⁺. Our results support the hypothesis that an anionic current that occurs during SR Ca²⁺ uptake is reduced by the substitution of Br or I for Cl.