Aminoglycoside antibiotics were found for the first time to inhibit an HIV- 1 Tat-derived peptide binding to TAR RNA. The ICso values of neomycin, streptomycin, and gentamicin were determined as 0.92 I.tM, 9.5 ~aM, and 45 I.tM, respectively. In the absence of Tat peptide these antibiotics were found to cause mobility shifts of the TAR RNA on non-denaturing polyacrylamide gels. This is the first example, to our knowledge, of non-peptide or non-nucleotide like small molecules that bind to and induce a mobility shift of TAR RNA. It was further found that the antibiotics which demonstrate higher affinity for TAR are better inhibitors of the Tat/TAR interaction. Mutational and competition studies indicate that neomycin binds to the duplex domain of TAR RNA.