A total of 82 human immunodeficiency virus (HIV)-1-specific cytolytic T lymphocyte (CTL) clones were isolated and characterized from 5 HIV-infected subjects, utilizing multiple HLA class I alleles. B62-restricted, HIV-1 gag-specific CTL clones isolated from a single blood sample from 1 subject used four different Vbeta gene rearrangements. Multiple CTL clones could be isolated from the same time point directed against HIV-1 gag, nef, and env from 1 subject. A prospective analysis resulted in the isolation of CTL clones from 1 subject directed against multiple HIV-1 antigens, including the same highly conserved nef peptide, over a 1-year period, in the absence of detectable circulating viral plasma RNA. These data suggest that in some persons without clinical progression and low levels of circulating HIV-1, the CTL response is polyclonal, is directed against multiple HIV-1 proteins, including highly conserved peptides within these proteins, and is maintained over time.