AbstractThe human immunodeficiency virus type 1 (HIV‐1) integrase (IN) protein plays an important role during the early stages of the retroviral life cycle and therefore is an attractive target for therapeutic intervention. We immunized rabbits with HIV‐1 IN protein and developed a combinatorial single‐chain variable fragment (scFv) library against IN. Five different scFv antibodies with high binding activity and specificity for IN were identified. These scFvs recognize the catalytic and C‐terminal domains of IN and block the strand‐transfer process. Cells expressing anti‐IN–scFvs were highly resistant to HIV‐1 replication due to an inhibition of the integration process itself. These results provide proof‐of‐concept that rabbit anti‐IN–scFv intrabodies can be designed to block the early stages of HIV‐1 replication without causing cellular toxicity. Therefore, these anti‐IN–scFvs may be useful agents for “intracellular immunization”‐based gene therapy strategies. Furthermore, because of their epitope binding characteristics, these scFvs can be used also as new tools to study the structure and function of HIV‐1 IN protein.