The deoxythymidine-5'-triphosphate (dTTP) analog 3'-fluorothymidine 5'-triphosphate (3'-FdTTP) inhibits DNA synthesis by T4 wild-type, L98 (mutator) and CB121 (antimutator) DNA polymerase. CB121 DNA polymerase is less sensitive by a factor of two than the L98 and T4+ enzymes. Inhibition is not due to incorporation of the analog into DNA. 3'-FdTTP acts competitively to the substrate dTTP. The CB121 polymerase exhibits a higher Ki to Km ratio than the other two enzymes (5.3 vs. 3.3) and thus discriminates better between the substrate dTTP and its analog 3'-FdTTP. 3'-FdTTP inhibits the polymerase-associated 3'-5' exonuclease activities to the same extent as their polymerase activities. The CB121 3'-5' exonuclease activity is suppressed only half as much by 3'-FdTTP as by dTTP. The results are discussed in relation to the role of T4 DNA polymerase and its associated 3'-5' exonuclease in determining the accuracy of DNA replication.