Forty-four rifamycin derivatives with substitutions at the 3-carbon and 4-carbon positions of the rifamycin nucleus were tested for Inhibition of 1) DNA polymerases α, β, and γ (DPα, DPβ, and DPγ, respectively) from leukemia Iymphoblasts and phytohemagglutinin-stimulated normal human lymphocytes, 2) terminal deoxynucleotldyl transferase (tdT) from leukemia Iymphoblasts, and 3) reverse transcrlptase (RT) from simian sarcoma virus. Eighteen compounds were substantially Inhibitory to different degrees. One of these, 3'-acetyl-1'-benzyl-2'-methylpyrrolo[3,2-c]-4-desoxy-rlfamycln SV (derivative PRI19), selectively Inhibited TdT, and the other 17 compounds exhibited no striking selectivity toward any of the enzymes when assayed with the use of reaction mixtures containing no added bovine serum albumin (BSA). The structure-activity relationships of these results showed that compounds with long alkyl chains (8–11 carbon atoms) at the 3-carbon position of the rifamycin nucleus were among the most potent Inhibitors. Also, the addiction of aromatic moieties to side groups at the 3-carbon position could sometimes enhance the Inhibitory potential of a series of compounds. Inhibition of DPα, DPβ, and DPγ by rifamycin derivatives was completely or partially prevented by addition of BSA to assay mixtures at concentrations of 250 μg/ml, but inhibition of TdT and RT was unaffected by It. These data suggest a greater specificity of rifamycin derivatives for TdT and RT than for DPα, DPβ, and DPγ. Furthermore, a greater specificity of rifamycin derivatives for DPα, DPβ, and DPγ than for BSA was Indicated, Inasmuch as Inhibition of those DNA polymerases by rifamycin derivatives was prevented by concentrations of BSA (250 μg/ml) that were much higher than those of enzyme protein (≤3 μg/ml).