The Mono Q-III fraction, a Mg2(+)-ATPase, isolated from Acetabularia acetabulum was reconstituted into liposomes of various net charges prepared by the reversed-phase method and tested for a Cl(-)-translocating activity. The liposomes from a mixture of egg lecithin, dicetyl phosphate, and cholesterol (63:18:9 mole ratio, negative liposomes) and from a mixture of egg lecithin and cholesterol (63:9 mole ratio, neutral liposomes) were less leaky than positive liposomes from asolectin, and from a mixture of egg lecithin, stearylamine, and cholesterol (63:18:9 mole ratio). A significant increase in 36Cl- efflux from the negative and neutral liposomes was observed by addition of ATP in the presence of valinomycin after incorporation of the enzyme by short-term dialysis. The ATP-driven 36Cl- efflux was inhibited by addition of azide, an inhibitor of the ATPase. The preincubation of the enzyme with phenylglyoxal, an arginine-modifying reagent, inactivated ATP-mediated 36Cl- efflux, but the ATPase activity of the preparation was not affected. When chloride was replaced by 35SO4(2)-, no ATP-dependent 35SO4(2)- efflux was detectable from the proteoliposomes. Proton-translocating activity of the enzyme was also tested, and no fluorescent quenching of 9-ACMA was observed.