The transport of inorganic phosphate has been studied in Acinetobacter lwoffi JW11. During growth on excess phosphate, only one transport system was present, with an apparent K(m) of 1.4-mu-M. When cells-were starved for phosphate, a second uptake system with an apparent K(m) of 110 nM was also synthesized. The two transport systems could be distinguished by differing sensitivities to the phosphate analogs arsenate and 2-aminoethylphosphonate. Both systems were inhibited by carbonylcyanide m-chlorophenylhydrazone, and to a lesser extent by Na azide. The high-affinity transport system was inactivated by osmotic shock treatment and by spheroplast formation. Preliminary evidence for a phosphate-binding protein in the osmotic shock fluid is presented. The isolation of a mutant constitutive for the high-affinity transport system is described.