Guanylate kinase catalyzes the phosphorylation of either GMP to GDP or dGMP to dGDP and is an essential enzyme in nucleotide metabolism pathways. Despite its involvement in antiviral drug activation in humans and in mouse model systems and as a target for chemotherapy, the human and mouse primary structures have never been elucidated. Full-length cDNA clones encoding enzymatically active guanylate kinase were isolated from mouse B-cell lymphoma and human peripheral blood lymphocyte cDNA libraries. Multiple tissue Northern blots demonstrated an mRNA species of approximately 1 kilobase for both mice and humans in all tissue types examined. The mouse cDNA is predicted to encode a 198-amino acid protein with a molecular mass of 21,904 daltons. The human cDNA is predicted to encode a 197-amino acid protein with a molecular mass of 21,696 daltons. These proteins share 88% sequence identity with each other and 52-54% identity with the yeast guanylate kinase. Molecular modeling using the yeast diffraction coordinates indicates a high degree of conservation within the active site and maintenance of the overall structural integrity, despite a lack of similarity along the periphery of the enzyme.