BuPdGTP, the 2′-deoxyribonucleoside 5′-triphosphate of the DNA polymerase alpha (pol α)-specific inhibitor, N ² -(p-n-butylphenyl) guanine, was examined with respect to its mechanism and its capacity to inhibit the mammalian DNA polymerases, pol α, pol α, and pol γ. BuPdGTP was specifically inhibitory for pol α, with no discernible activity on pol β and pol γ. The potency of BuPdGTP is unprecedented, with an apparent K _i less than 10 nanomolar. The unusual potency of the BuPdGTP is derived primarily from the 5′ α and β phosphoryl moieties, whose binding to enzyme complements that of the base-linked butylphenyl substituent. BuPdGTP is competitive with dGTP and apparently not subject to polymerization. Experiments employing BuPdGTP in the presence of a non-complementary template suggest that the core polymerase or an associated coprotein contains dNTP binding sites which recognize specific nucleic acid bases. The partial sensitivity of selected, non-mammalian DNA polymerases suggests that modification of the N ² substituent of dGTP will be a useful route to the design of novel, polymerase-specific affinity-probes.