We studied the Ca(2+)-ATPase isoforms of sarco/endoplasmic reticulum (SERCA) derived from cerebellum, cardiac muscle, and skeletal muscle. The Mg2+ dependence varied among the three enzyme preparations. The Ca2+ transport in skeletal muscle vesicles, but not in cerebellar or cardiac vesicles, was activated by free Mg2+ concentrations varying from 0.1 to 0.3 mM. Concentrations of Mg2+ of > 1 mM inhibited Ca2+ transport in all three vesicle preparations but with more pronounced effect in cerebellar and cardiac vesicles. At 10-80 microM, trifluoperazine activated Ca2+ uptake in cerebellar and cardiac vesicles but not in skeletal muscle vesicles. The activation was due to an increase in the coupling ratio between Ca2+ transport and ATP hydrolysis and was observed only in the presence of ATP concentrations of > 100 microM. The Ca2+ transport in all three vesicle preparations was inhibited by trifluoperazine concentrations of > 100 microM. The inhibition promoted by trifluoperazine was prevented by the addition of dimethylsulfoxide (10% v/v) to the medium. The Ca2+ efflux from loaded vesicles was increased by arsenate and even more by trifluoperazine. In skeletal muscle vesicles, the efflux promoted by arsenate was several-fold faster than that promoted in vesicles derived from cerebellum or cardiac muscle. In skeletal muscle, the enhancement of Ca2+ efflux promoted by both arsenate and trifluoperazine was antagonized by thapsigargin, Ca2+, Mg2+, and K+. These agents partly antagonized the enhancement of Ca2+ efflux promoted by trifluoperazine in cardiac vesicles but had little or no effect in the cerebellar vesicles. Finally, Mg.Pi and Mg.ATP, the two substrates that phosphorylate the Ca(2+)-ATPase, antagonized the effect of trifluoperazine in all of the preparations tested. The concentration of ATP needed was in the same range as that of the second K(m) value for ATP (50-300 microM) of the SERCA isoforms. The results indicate that the effect of the drugs on the cytosolic Ca2+ homeostasis may vary depending on the target tissue.