The ATP-generating system described by Hugenholtz, J., Hong, J.-S. and Kaback, H.R. ((1981) Proc. Natl. Acad. Sci. USA 78, 3446-3449) has been used to synthesize ATP up to 1.8 mM in right-side-out membrane vesicles from Escherichia coli. This ATP level was sufficient to drive uptake of potassium ions via the Kdp-ATPase. In the kdp wild type strain about 110 nmoles K+/mg membrane protein were accumulated. This process was still partially sensitive to the well-known inhibitors of P-type ATPases, orthovanadate and bafilomycin B1.