Partly purified membranes (with plasmalemma material) of Acetabularia mediterranea were studied with respect to ATPase activity in alkali- and Ca(++)-free media and its sensitivity to pH (5 - 9), oligomycin (200 ώg/mg protein), 100 ώM N-N'-dicyclohexylcarbodiimide (DCCD), and 50 ώM vanadate. Besides activities which may originate from mitochondrial H(+) ATPase (oligomycin-sensitive, alkaline pH optimum) and tonoplast H(+) ATPase (DCCD-sensitive, pH optimum 7.5), there is ATPase activity with a pH optimum around pH 6.5, sensitive to vanadate and insensitive to DCCD. These results strongly suggest that the electrogenic Cl(-) pump in the plasmalemma of Acetabularia is an ATPase. Effects of Mg(++), Mg-ATP, ADP, GTP, UTP, CTP and HCO3 (-) versus Cl(-) on this ATPase activity are described.