In permeabilized parotid cells and in isolated membrane vesicles from parotid endoplasmic reticulum (ER), Mg-ATP-dependent Ca2+ uptake was measured using a Ca2+-specific macroelectrode and 45Ca2+, respectively. Mg-ATP-dependent Ca2+ uptake was inhibited by vanadate (2 x 10(-3) mol/l) by approximately 45% in permeabilized cells and by approximately 70% in membrane vesicles from ER during the initial 10 min. After this lag phase, Ca2+ uptake increased and low steady-state free [Ca2+] of approximately 3 x 10(-7) mol/l was still reached in presence of vanadate within 30-40 min. Subsequent addition of inositol 1,4,5-trisphosphate (IP3) caused a similar Ca2+ release compared with control. This indicates that in presence of vanadate an IP3-sensitive Ca2+ pool was filled. However, when protonophores, such as nigericin or carbonyl cyanide-m-chlorophenylhydrazone, were added in addition to vanadate, this low steady-state free [Ca2+] was not reached. 45Ca2+ uptake was reduced by approximately 70% within 60 min, and IP3 did not cause 45Ca2+ release when given subsequently, indicating that filling of an IP3-sensitive Ca2+ pool was prevented. Mg-ATP-driven H+ uptake into ER vesicles, as estimated with acridine orange, was abolished by protonophores and by the H+-ATPase blockers N-ethylmaleimide and Dio 9 but was unaltered by vanadate. Preincubation of ER vesicles in a medium without Ca2+, but with vanadate and with Mg-ATP to generate an H+ gradient, allowed demonstration of 45Ca2+ uptake from a medium that did not contain ATP. The cation sequence in absence of vanadate for support of Mg-ATP-dependent 45Ca2+ uptake was K+ greater than Na+ greater than Li+ = choline+ and, in presence of vanadate, was choline+ greater than Li+ = Na+ greater than K+. A preformed H+ gradient dissipated more rapidly in presence of K+ compared with choline+, probably due to an intrinsic K+ permeability of ER membrane. Our data indicate that both a Ca2+ and a H+ pump are located in a compartment of ER that is also sensitive to IP3. Ca2+ uptake is coupled to an H+ gradient that is generated by the H+ pump and most likely occurs via Mg-ATP-driven Ca2+-H+ countertransport but to some extent can also operate in absence of ATP at the expense of the H+ gradient.