In order to elucidate the biological activities of cordycepin (3'-deoxyadenosine) and related 3'-deoxyribonucleosides on eukaryotic DNA replication, the inhibitory effects of triphosphate derivatives of 3'-deoxyadenosine(3'-dATP), 8-azido-3'-deoxyadenosine(8-N3-3'-dATP), 3'-deoxyguanosine(3'-dGTP), 3'deoxyuridine(3'dUTP), 5-fluoro-3'deoxyuridine(5-F-3'-dUTP), 3'-deoxycytidine(3'-dcTP), and 5-fluoro-3'-deoxycytidine(5-F-3'dCTP) on DNA primase and replicative DNA polymerases alpha, delta, and epsilon purified from cherry salmon (Oncorhynchus masou) testes or calf thymus were examined. All analogs, except 8-N3-3'-dATP, showed strong inhibitory effects on DNA primase, but none of them inhibited DNA polymerases alpha, delta, or epsilon. Kinetic analysis revealed that the inhibition modes of them were competitive with respect to the incorporation of natural substrate that had the corresponding base moiety and non-competitive with respect to other substrates. Based on the kinetic data, we compared the affinities of 3'-dNTPs between DNA primase and RNA polymerases I and II, since 3'-dNTPs also inhibit eukaryotic RNA polymerases. Although the Ki values for DNA primase were much larger than those for RNA polymerases, the Ki/K(m) values, which indicate the affinity of the analog to the enzyme, were very similar.