The electrogenic Cl(-)-pump in the plasmalemma of the marine alga Acetabularia acetabulum (L.) Silva has been suggested to be an unusual type of ATPase (Gradmann, 1989, Methods Enzymol. 174, 490). For a biochemical treatment of this issue, a plasmalemma-rich membrane fraction from Acetabularia has been prepared by phase-partitioning. About 80% of the ATPase activity in this material is inhibited by vanadate (K I50=1-2 μM). The phosphohydrolytic properties of the corresponding enzyme were further investigated. Its primary substrate MgATP(2-) (K m about 270 μM). Compared with other plasmalemma ATPases, it has an extremely alkaline pH optimum (pH 8-8.5), a weak sensitivity to diethyl-stilbestrol and to N,N'-dicyclohexylcarbodiimide, a strong selectivity for Mg(2+) over alternative divalent cations, and a weak selectivity for ATP over other phosphohydrolytic substrates. It is insensitive to KCl at concentrations up to 200 mM. Both ATP(4-) and Mg2ATP inhibit the ATPase, satisfying a relationship for competitive inhibition by 2ATP(4-) (K IATP=1.56 mM) and noncompetitive inhibition by Mg2ATP (K IMg2ATP=1.35 mM). Since no transport experiments are reported in this study, the ion species (H(+) or Cl(-)) that is transferred by this ATPase is not identified.