We have investigated phosphorylation of human nucleoside diphosphate kinase (NDPK) and of homologous NDPK from different species by human casein kinase 2 (CK-2). The human NDPK isotypes A and B were phosphorylated by CK-2 in vitro both when the purified proteins and total lysate of HL-60 leukemia cells were used. The homologous NDPK's from Yeast and E. coli were also substrates for CK-2 in vitro, but not Drosophila NDPK. Phosphorylation of all NDPK types by the CK-2 holoenzyme was entirely polyamine-dependent. The CK-2 phosphorylation site in human NDPK A, that was about 2.5 times stronger phosphorylated than was the B isotype, was tentatively assigned to Ser-122. The location of the corresponding residue in the 3D-structure of the 80% homologous Drosophila NDPK suggests that its phosphorylation may directly influence substrate binding and/or catalysis.