In this study we investigated 13 enzymes of T. maritima for their ability to phosphorylate natural and modified nucleosides or nucleotides. Three of the enzymes, namely TmAsK, TmUK and TmNuc, did not show phosphotransferase activity with ATP as phosphate donor under the conditions tested. Broad substrate spectra towards natural substrates were shown for TmTK, TmPK and TmAcK, whereas TmPPDK and TmRK were more specific. While all five TmNMPKs had a narrow substrate scope towards NMPs, we detected a broad NDPK activity for three TmNMPKs. In addition, we demonstrated for the first time the phosphorylation of base-and sugarmodified NMPs by thermostable NMPKs in enzymatic cascade reactions. Thus, our work lays the foundation for the application of these thermostable enzymes for the biocatalytic production of nucleotide analogs.