Agrobacterium tumefacienssynthesizes polyphosphate (polyP) in the form of one or two polyP granules per cell during growth. TheA. tumefaciensgenome codes for two polyphosphate kinase genes,ppk1(AT)andppk2(AT), of which onlyppk1(AT)is essential for polyP granule formation in vivo. Biochemical characterization of the purified PPK1(AT)and PPK2(AT)proteins revealed a higher substrate specificity of PPK1(AT)(in particular for adenine nucleotides) than for PPK2(AT). In contrast, PPK2(AT)accepted all nucleotides at comparable rates. Most interestingly, PPK2(AT)catalyzed also the formation of tetra-, penta-, hexa-, hepta-, and octa-phosphorylated nucleosides from guanine, cytosine, desoxy-thymidine, and uridine nucleotides and even nona-phosphorylated adenosine. Our data-in combination with in vivo results-suggest that PPK1(AT)is important for the formation of polyP whereas PPK2(AT)has the function to replenish nucleoside triphosphate pools during times of enhanced demand. The potential physiological function(s) of the detected oligophosphorylated nucleotides await clarification.