Partially 5-thiolated polyuridylic acid (poly(U-60,hs(5)U(40))) is shown to be a potent inhibitor of Moloney murine leukemia virus Reverse Transcriptase (M-MuLV RT). The pattern of this inhibition is competitive, when wither poly(A).(dT)(16) or poly(D).(dG)(16) as template-primer (variable substrate) are used, suggesting that the free enzyme interacts with the modified polynucleotide. K-m and K-i values of 25 mu M and 11 nM, respectively, were obtained in the presence of poly(A).(dT)(16). The K-i value determined in the presence of poly(C).(dG)(16) was 31 nM (K-m = 22 mu M). Inhibitory activities of the 5-thiolated oligouridylic acids, prepared from the polymer, depend on the chain-length. While the 30-mer showed the same activity as the intact polynucleotide, shorter oligonucleotides proved to be less active.