Commercial-grade aurintricarboxylic acid (ATA) inhibits poly(A), poly(C) and viral RNA-directed DNA synthesis by detergent-disrupted virions of Moloney murine leukemia virus. Paper chromatography of crude ATA yields two active components, which appear to behave identically, and at least two inactive components. The concentration of ATA needed to inhibit polymerase activity is proportional to the concentration of viral protein. The inhibition is neither attributable to contaminating heavy metal ions in the ATA preparation nor to chelation by ATA of Mn2+ or Zn2+, the necessary co-factors. Inhibition of the polymerase reaction by ATA greatly increases the Km for the primer [oligo(T)/oligo(dG)], while it only slightly lowers the Vmax and does not affect the Km's for the template [poly(A)/poly(C)] or the substrate (TTP/dGTP). Thus, ATA seems to reduce specifically the affinity of the polymerase for the DNA primer molecule.