We produced a series of monoclonal antibodies against the human immunodeficiency virus (HIV-1) reverse transcriptase by immunizing mice with either purified recombinant HIV-1 p66 protein or with recombinant vaccinia virus which expresses HIV-1 pol sequences. The antibodies generated were specific for the reverse transcriptase protein, and recognized only the p51 and p66 subunits of the enzyme in each of the HIV-1 viral lysates and lysates of HIV-1 infected cells. The antibodies did not cross-react with HIV-2 reverse transcriptase. Most important, several of the antibodies are unique, in that they are the first that can bind to sites close to the N-terminal. This latter region has been suggested to form part of the polymerase domain of the reverse transcriptase. None of the antibodies could neutralize either the RNA-dependent DNA polymerase or RNase H activities of either p66 or p51/66 proteins. The binding patterns of these various antibodies to p66 and p51/66 were dependent on each of three independent variables: the source of antigen amployed, the individual specificity of the antibody, and the method employed to detect reactivity. These monoclonal antibodies provide useful reagents for the study of reverse transcriptase native structure-function relationships.