Inhibitory antibodies to the reverse transcriptase (RT) of human immunodeficiency virus type-1 (HIV-1) can be used to block the life cycle of the virus. We have isolated five different human single chain Fv (ScFv) antibodies specific for HIV-1 RT from an antibody phage display library. Three of these antibodies inhibited the RNA-dependent DNA polymerase (RDDP) activity of RT and one of the three (F-6) inhibited also its DNA-dependent DNA polymerase (DDDP) activity. Unexpectedly, F-6 binds to the carboxyl terminus of the large subunit of RT, which contains the ribonuclease H (RNase H) domain, and not the polymerase domain of the protein. Moreover, this binding did not inhibit the RNase H enzymatic activity. To further characterize F-6 antibody, two cyclic synthetic peptides based on the amino acids sequences of the CDR3 of F-6 were synthesized. Peptide F-6CDRH3, with the sequence of CDR3 of the heavy chain, inhibited the RDDP activity of RT while peptide F-6CDRL3, with the sequence of CDR3 of the light chain, had no effect on this activity of RT. These results indicate that some of the effects of F-6 are mediated by the CDR3 of the heavy chain. The antibodies identified here will be further tested as intrabodies for their capacity to protect human cells from HIV-1 infection.