CD8(+) lymphocytes from human immunodeficiency virus (HIV)-infected patients can suppress in vitro HIV replication in CD4(+) T cells by a noncytolytic mechanism involving secreted CD8(+)-cell antiviral factor(s) (CAF). Using an HIV Nef-specific cytotoxic-T-lymphocyte (CTL) line and autologous CD4(+) T cells infected with a nef-deleted HIV-1 virus, we demonstrated that, after a priming antigenic stimulation, this suppression does not require the presence of the specific antigen during the effector phase. Furthermore, using an Epstein-Barr virus (EBV)-specific CTL line from an HIV-seronegative donor, we demonstrated that the ability to inhibit HIV replication in a noncytolytic manner is not restricted to HIV-specific effector cells; indeed, EBV-specific CTL were as efficient as HIV-specific effectors in suppressing R5 or X4 HIV-1 strain replication in vitro. This HIV-suppressive activity mediated by a soluble factor(s) present in the culture supernatant was detectable for up to 14 days following stimulation of EBV-specific CD8(+) cells with the cognate epitope peptide. Following acute infection of CEM cells with an X4 strain of HIV-1, EBV-specific CTL line supernatant containing HIV-suppressive activity did not block virus entry but was shown to interfere with virus replication after the first template switching of reverse transcription. Our results suggest that the noncytolytic control of HIV replication by EBV-specific CD8(+) T lymphocytes corresponded to a CAF-like activity and thus demonstrate that CAF production may not be restricted to CTL induced during HIV disease. Moreover, CAF acts after reverse transcription at least for X4 isolate replication inhibition.