Phylogenetic analysis of viral and cellular thymidine and thymidylate kinases was performed using computer-assisted methods. Multiple alignments and tentative phylogenetic trees were generated for the two families of these enzymes, which include a) thymidine kinases (TK) of mammals, poxviruses, African swine fever virus, E. coli, and bacteriophage T4; and b) thymidylate kinases (ThyK) of yeast and poxviruses and distantly related herpesvirus proteins with both enzymatic activities. Analysis of the alignment of the TKs of the first family highlighted three strongly conserved segments. Two of these corresponded to the A and B motifs of the purine NTP-binding pattern. The third, C-terminal segment, showing the highest conservation, encompassed a modified Zn finger motif. It is speculated that this motif might be involved in TK oligomerization. Phylogenetic trees constructed by three different methods suggested that cellular TK genes could be captured independently by T4 bacteriophage, African swine fever virus, fowlpox virus, and the other poxviruses. The observed tree topologies appear to contradict the popular virus-host coevolution schemes and to imply that different subdivisions of poxviruses diverged at earlier stages of evolution than their hosts did. It was shown that deoxynucleoside monophosphate kinase of bacteriophage T4 is related to the ThyK family. Phylogenetic analysis suggested that ThyK genes probably have been acquired independently by phage T4, poxviruses, and herpes-viruses.