The determinants of CD8⁺ cytotoxic T-lymphocyte (CTL) antiviral activity against human immunodeficiency virus type 1 (HIV-1) remain poorly defined. Although recent technological advances have markedly enhanced the ability to detect HIV-1-specific T cells, commonly used assays do not reveal their direct interaction with virus. We investigated two determinants of CTL antiviral efficiency by manipulating HIV-1 and measuring the effects on CTL suppression of viral replication in acutely infected cells. Translocation of a Gag epitope into the early protein Nef markedly increased the activity of CTL recognizing that epitope, in comparison to HIV-1 expressing the epitope normally in the late protein Gag. Because this epitope translocation resulted not only in earlier expression but also in loss of major histocompatibility complex class I downregulation by Nef, the activities of CTL against a panel of viral constructs differing in kinetics of epitope expression and class I downmodulation were compared. The results indicated that both the timing of epitope expression and the reduction of class I have profound effects on the ability of CTL to suppress HIV-1 replication in acutely infected cells. The epitope targeting of CTL and viral control of class I therefore likely play important roles in the ability of CTL to exert pressure on HIV-1.