Despite the fact that Ca2+ transport into the sarcoplasmic reticulum (SR) of muscle cells is electrogenic, a potential difference is not maintained across the SR membrane. To achieve electroneutrality, compensatory charge movement must occur during Ca2+ uptake. To examine the role of Cl- in this charge movement in smooth muscle cells, Ca2+ transport into the SR of saponin-permeabilized smooth muscle cells was measured in the presence of various Cl- channel blockers or when I-, Br-, or SO42- was substituted for Cl-. Calcium uptake was inhibited in a dose-dependent manner by 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) and by indanyloxyacetic acid 94 (R(+)-IAA-94), but not by niflumic acid or 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS). Smooth muscle SR Ca2+ uptake was also partially inhibited by the substitution of SO42- for Cl-, but not when Cl- was replaced by I- or Br-. Neither NPPB nor R(+)-IAA-94 inhibited Ca2+ uptake into cardiac muscle SR vesicles at concentrations that maximally inhibited uptake in smooth muscle cells. These results indicate that Cl- movement is important for charge compensation in smooth muscle cells and that the Cl- channel or channels involved are different in smooth and cardiac muscle cells.