Thymidine triphosphate (TTP) and its halogenated analog bromodeoxyuridine triphosphate (BrdUTP) were compared in vitro as substrates for several prokaryotic and eukaryotic DNA polymerases to determine a possible enzymatic preference which might account for the reported finding of nonrandom patterns of incorporation of the analog in eukaryotic cellular DNA as well as help clarify the mechanism for drug-induced activation of latent retroviruses from animal cells. Following nucleotide competition reactions, no discriminatory utilization was detected from a mixture containing equimolar [3H]TTP and [alpha 32P]TTP for any of the polymerases. In contrast, when [3H]BrdUTP was mixed with an equal concentration of [alpha 32P]TTP, it was apparent that eukaryotic DNA polymerase gamma utilized more of the brominated analog triphosphate in preference to the unsubstituted compound. This increased affinity was confirmed by the differences in Km values. Furthermore, the selectivity of polymerase gamma was even more pronounced with the iodinated thymidine analog iododeoxyuridine triphosphate. On the other hand, polymerase gamma failed to discriminate as readily between equal concentrations of alpha 32P-labeled deoxycytidine triphosphate and 125I-labeled iododeoxycytidine triphosphate.