The enzyme adenosine kinase constitutes the major purine nucleoside phosphorylating activity in mammalian cells. In view of its central role in adenosine metabolism, which is an important physiological regulator, an understanding of the primary structure of adenosine kinase is of much interest. Using microsequence information from peptides derived from purified Syrian hamster liver enzyme, we have succeeded in isolating full length cDNA clones encoding adenosine kinase from Chinese hamster ovary cells and mouse 3T3 cells. The open reading frames in these clones consist of 334 and 335 amino acids and encode proteins of molecular masses 37364 Da and 37489 Da, respectively. In addition, the coding and upstream sequences for adenosine kinase from human (HeLa cells) and rat liver have also been cloned and sequenced. Transfection of an adenosine-kinase-deficient mutant (selected for resistance to the adenosine analog toyocamycin) of Chinese hamster ovary cells with a plasmid containing the cloned adenosine kinase cDNA, leads to regaining of adenosine kinase activity in the transformed cell. The adenosine kinase transformants also simultaneously lost their toyocamycin resistance and became similarly sensitive to the analog as the parental wild-type Chinese hamster ovary cells. The cloned adenosine kinase cDNA was also used to examine structural changes in mutants affected in adenosine kinase. In Chinese hamster ovary cells, one type of mutant that lacks adenosine kinase activity and displays high degree of resistance to various adenosine analogs, is obtained at an unusually high spontaneous frequency (10(-4)-10(-3)). Results of Southern and northern-blot analysis provide evidence that this group of mutants involves gross structural alterations affecting the adenosine kinase gene. Such structural alterations are not observed in another type of mutant which exhibits increased resistance only to C-adenosine analogs. Sequence similarity searches indicate that several of the bacterial and yeast sugar kinases (ribokinase, fructokinase and inosine-guanosine kinase) exhibit limited but significant similarity to the mammalian adenosine kinase. The sequence similarity data support the possibility that adenosine kinase shares a common evolutionary ancestor with these protein sequences.