Strains of Acinetobacter which showed marked variation in their ability to accumulate intracellular polyphosphate (Pn) were isolated from a pilot-plant which was removing phosphate biologically. Variants which could not accumulate Pn under the same growth conditions were derived from two of the isolates which accumulated high levels of Pn. The activities of five enzymes reported to have a role in Pn synthesis showed no significant differences between the two variants, their parent strains and two other natural isolates. In the presence of 20 μm N,N'-dicyclohexylcarbodiimide (DCCD), growth of the variant strains was suppressed, whereas the parent strains were still able to grow and form polyphosphate. A mechanism which depends upon the trans-membrane proton gradient of the cell is proposed to account for the high levels of polyphosphate formed by some strains of Acinetobacter. Each of the Acinetobacter strains isolated from the pilot-plant carried several plasmids. Comparison of one strain, which accumulated a high level of Pn, and its variant, showed that a deletion of approximately 20 kb of plasmid DNA from the parent strain had occurred. Strains of Acinetobacter which showed marked variation in their ability to accumulate intracellular polyphosphate (Pn) were isolated from a pilot-plant which was removing phosphate biologically. Variants which could not accumulate Pn under the same growth conditions were derived from two of the isolates which accumulated high levels of Pn. The activities of five enzymes reported to have a role in Pn synthesis showed no significant differences between the two variants, their parent strains and two other natural isolates. In the presence of 20 μm N,N′-dicyclohexylcarbodiimide (DCCD), growth of the variant strains was suppressed, whereas the parent strains were still able to grow and form polyphosphate. A mechanism which depends upon the trans-membrane proton gradient of the cell is proposed to account for the high levels of polyphosphate formed by some strains of Acinetobacter. Each of the Acinetobacter strains isolated from the pilot-plant carried several plasmids. Comparison of one strain, which accumulated a high level of Pn, and its variant, showed that a deletion of approximately 20 kb of plasmid DNA from the parent strain had occurred.