In acetate-limited chemostat cultures of Acinetobacter johnsonii 210A at a dilution rate of 0.1 h-1 the polyphosphate content of the cells increased from 13% to 24% of the biomass dry weight by glucose (100 mM), which was only oxidized to gluconic acid. At this dilution rate, only about 17% of the energy from glucose oxidation was calculated to be used for polyphosphate synthesis, the remaining 83% being used for biomass formation. Suspensions of non-growing, phosphate-deficient cells had a six- to tenfold increased uptake rate of phosphate and accumulated polyphosphate aerobically up to 53% of the biomass dry weight when supplied with only orthophosphate and Mg2+. The initial polyphosphate synthesis rate was 98 ± 17 nmol phosphate min-1 mg protein-1. Intracellular poly-β-hydroxybutyrate and lipids served as energy sources for the active uptake of phosphate and its subsequent sequestration to polyphosphate. The H+-ATPase inhibitor N,N'- dicyclohexylcarbodiimide caused low ATP levels and a severe inhibition of polyphosphate formation, suggesting the involvement of polyphosphate kinase in polyphosphate synthesis. It is concluded that, in A. johnsonii 210A, (i) polyphosphate is accumulated as the energy supply is in excess of that required for biosynthesis, (ii) not only intracellular poly-β- hydroxybutyrate but also neutral lipids can serve as an energy source for polyphosphate-kinase-mediated polyphosphate formation, (iii) phosphate- deficient cells may accumulate as much polyphosphate as activated sludges and recombinants of Escherichia coli designed for polyphosphate accumulation.