Tonoplast-and plasma membrane-enriched fractions were simultaneously prepared from the same crude membrane suspension from spinach leaves by free-flow elec~rophoresis. They were used for in vitro ATP-dependent Ca 2 +-uptake studies. The plasma membrane-enriched fraction accumulated Ca 2 + at a rather slow rate by a mechanism which was inhibited by orthovanadate, insensitive to nitrate and activated by calmodulin. The tonoplast-enriched fraction accumulated Ca 2 ÷ at a high rate for 5 rain, then the accumulated Ca 2 + progressively leaked out of the vesicles. The tonoplast transport system was partly inhibited by nitrate, but also by vanadate, completely abolished by the protonophore CCCP and activated by caimodulin and the Ca 2 + antagonist verapamil.