Two active calcium (Ca(2+)) transport systems have been identified and partially characterized in membrane vesicles isolated from cultured carrot cells (Daucus carota Danvers). Both transport systems required MgATP for activity and were enhanced by 10 millimolar oxalate. Ca(2+) transport in membrane vesicles derived from isolated vacuoles equilibrated at 1.10 grams per cubic centimeter and comigrated with Cl(-)-stimulated, NO(3) (-)-inhibited ATPase activity on sucrose density gradients. Ca(2+) transport in this system was insensitive to vanadate, but was inhibited by nitrate, carbonyl cyanide-m-chlorophenylhydrazone (CCCP), N,N'-dicyclohexylcarbodiimide (DCCD), and 4,4-diisothiocyano-2,2'-stilbene disulfonic acid (DIDS). The K(m) for MgATP and Ca(2+) were 0.1 mm and 21 micromolar, respectively. The predominant Ca(2+) transport system detectable in microsomal membrane preparations equilibrated at a density of 1.13 grams per cubic centimeter and comigrated with the endoplasmic reticulum (ER) marker, antimycin A-insensitive NADH-dependent cytochrome c reductase. Ca(2+) transport activity and the ER marker also shifted in parallel in ER shifting experiments. This transport system was inhibited by vanadate (I(50) = 12 micromolar) and was insensitive to nitrate, CCCP, DCCD, and DIDS. Transport exhibited cooperative MgATP dependent kinetics. Ca(2+) dependent kinetics were complex with an apparent K(m) ranging from 0.7 to 2 micromolar. We conclude that the vacuolar-derived system is a Ca(2+)/H(+) antiport located on the tonoplast and that the microsomal transport system is a Ca,Mg-ATPase enriched on the ER. These two Ca(2+) transport systems are proposed to restore and maintain cytoplasmic Ca(2+) homeostasis under changing cellular and environmental conditions.