In order to clarify the biological activities of (-)-oxetanocin G, and (-)-oxetanocin A and its carbocyclic analogue, (-)-carboxetanocin G, the inhibitory effects of triphosphate derivatives of these compounds (OXT-GTP, OXT-ATP, and C-OXT-GTP) on eukaryotic and viral DNA polymerases were examined. DNA polymerase α purified from calf thymus was weakly inhibited by OXT-GTP and OXT-ATP but strongly by C-OXT-GTP, the K₁ value being 0.22μM. On the other hand, rat DNA polymerase β was not affected by these analogues. DNA polymerase γ purified from bovine testes was very weakly inhibited by OXT-GTP and OXT-ATP, but not by C-OXT-GTP. DNA polymerase from herpes simplex virus type-II (HSV-II) was strongly inhibited by all three analogues, the K₁ values ranging from 0.5 to 1.0μM. Human immunodeficiency virus-encoded reverse transcriptase (HIV RT) was also strongly inhibited by these three analogues, the K₁ value of C-OXT-GTP being slightly smaller than that of OXT-GTP or OXT-ATP. Analysis of products synthesized on singly primed M13 single-stranded DNA by DNA polymerase α, HSV-II DNA polymerase or HIV RT in the presence of the analogues revealed that OXT-GTP and C-OXT-GTP were incorporated into DNA and caused chain termination mainly at sites one or two nucleotides beyond the cytosine bases on the template.