The transport of Pi was characterized in Acinetobacter johnsonii 210A, which is able to accumulate an excessive amount of phosphate as polyphosphate (polyP) under aerobic conditions. P. is taken up against a concentration gradient by energy-dependent, carrier-mediated processes. A. johnsonii 210A, grown under P.limitation, contains two uptake systems with K, values of 0.7 ± 0.2 PuM and 9 ± 1 uM. Pi uptake via the high-affinity component is drastically reduced by NN'-dicyclohexylcarbodiimide, an inhibitor of H+-ATPase, and by osmotic shock. Together with the presence of P,-binding activity in concentrated periplasmic protein fractions, these results suggest that the high-affinity transport system belongs to the group of ATP-driven, binding-protein-dependent transport systems. Induction of this transport system upon transfer of cells grown in the presence of excess Pi to P,-free medium results in a 6-to 10-fold stimulation of the Pi uptake rate. The constitutive low-affinity uptake system for P1 is inhibited by uncouplers and can mediate counterflow of Pi, indicating its reversible, secondary nature. The presence of an inducible high-affinity uptake system for P. and the ability to decrease the free internal P1 pool by forming polyP enable A. johnsonii 210A to reduce the P1 concentration in the aerobic environment to micromolar levels. Under anaerobic conditions, polyP is degraded again and Pi is released via the low-affinity secondary transport system. Enhanced biological phosphorus removal from domestic wastewaters in full-scale activated-sludge plants is currently perceived to hinge on the provision of alternate stages in which the activated sludge is subjected to anaerobic and aerobic conditions (40). A characteristic feature of such plants is that Pi, after being released from the biomass in an anaerobic stage, is reincorporated in the biomass during aeration, together with part or all of the influent Pi (16).