The ability of specific virally encoded proteins to down-regulate MHC class I molecules may enable infected cells to elude killing by CTL. In the case of HIV-1, Nef appears to be responsible for this effect. Thus, interfering with Nef-induced MHC class I down-regulation would be a strategy for increasing HIV-1-specific CTL activity, particularly towards long-lived T cell populations such as memory T cells that harbor replication-competent virus. Here, using two Nef-expressing human cell model systems, we show that a dominant-negative mutant derived from the Hck protein-tyrosine kinase, composed of the Hck N-terminal region, as well as the SH3 and SH2 domains, was able to inhibit Nef-induced MHC class I molecule down-regulation. This effect was SH3 domain dependent as it was not evident when the cells were transfected with DN-Hck-W93F, an SH3 domain mutant. The inhibitory effect of dominant-negative-Hck (DN-Hck) on Nef-induced class I down-regulation suggests that this Nef-mediated effect requires an interaction between the Nef polyproline site and an SH3-containing cellular protein that is involved in MHC class I molecule turnover. Interfering with the function of the Nef SH3 binding site in this way represents a strategy for assisting the host CTL response to clear HIV-1-infected cells.