In an effort to expand the genetic alphabet, a number of unnatural, predominantly hydrophobic, nucleoside analogues have been developed which pair selectively in duplex DNA and during enzymatic synthesis. Significant progress has been made toward the efficient in vitro replication of DNA containing these base pairs. However, the in vivo expansion of the genetic alphabet will require that the unnatural nucleoside triphosphates be available within the cell at sufficient concentrations for DNA replication. We report our initial efforts toward the development of an unnatural in vivo nucleoside phosphorylation pathway that is based on nucleoside salvage enzymes. The first step of this pathway is catalyzed by the D. melanogaster nucleoside kinase, which catalyzes the phosphorylation of nucleosides to the corresponding monophosphates. We demonstrate that each unnatural nucleoside is phosphorylated with a rate that should be sufficient for the in vivo replication of DNA.