CD8 ؉ cells from healthy HIV-infected individuals can suppress HIV replication in infected CD4 ؉ cells without killing the cells. This CD8 ؉ cell noncytotoxic antiviral response (CNAR), observed by coculture of CD8 ؉ cells with infected CD4 ؉ cells, is associated with secretion of a CD8 ؉ cell antiviral factor (CAF). In attempts to identify CAF, we discovered that certain protease inhibitors, particularly leupeptin, can block, by up to 95%, the anti-HIV activity in CD8 ؉ cell culture fluids as well as inhibit CNAR. The effect is dose-dependent and is observed in up to 70% of the CAF and CNAR assays by using fluids and cells from several different subjects. Pretreatment of CD8 ؉ cells with leupeptin reduces CNAR, further supporting an inhibitory effect on a CD8 ؉ cell product. This inhibitory activity of protease inhibitors does not affect cell growth, expression of activation antigens, or viability of either CD8 ؉ cells or the infected CD4 ؉ cells. The results suggest that a part of the CD8 ؉ cell noncytotoxic response involves the activity of a protease or a protein that interacts with protease inhibitors. Proteolysis of a CD8 ؉ cell product(s) may be involved. This observation offers a promising approach for identifying the mechanism of CNAR͞CAF activity.