Partially purified plasma membrane fractions were prepared from guinea-pig pancreatic acini. These membrane preparations were found to contain an ATP-dependent Ca(2+)-transporter as well as a heterogenous ATP-hydrolytic activity. The Ca(2+)-transporter showed high affinity for Ca2+ (KCa2+ = 0.04 +/- 0.01 microM), an apparent requirement for Mg2+ and high substrate specificity. The major component of ATPase activity could be stimulated by either Ca2+ or Mg2+ but showed a low affinity for these cations. At low concentrations, Mg2+ appeared to inhibit the Ca(2+)-dependent ATPase activity expressed by these membranes. However, in the presence of high Mg2+ concentration (0.5-1 mM), a high affinity Ca(2+)-dependent ATPase activity was observed (KCa2+ = 0.08 +/- 0.02 microM). The hydrolytic activity showed little specificity towards ATP. Neither the Ca(2+)-transport nor high affinity Ca(2+)-ATPase activity were stimulated by calmodulin. The results demonstrate, in addition to a low affinity Ca2+ (or Mg2+)-ATPase activity, the presence of both a high affinity Ca(2+)-pump and high affinity Ca(2+)-dependent ATPase. However, the high affinity Ca(2+)-ATPase activity does not appear to be the biochemical expression of the Ca(2+)-pump.