Human immunodeficiency virus (HIV)'s tremendous sequence variability is a major obstacle for the development of cytotoxic-T-lymphocyte-based vaccines, especially since much of this variability is selected for by CD8(+) T cells. We investigated to what extent reactivity to escape variant peptides in standard enzyme-linked immunospot (ELISPOT) assays predicts the recognition of cells infected with corresponding escape variant viruses. Most of the variant peptides tested were recognized in standard ELISPOT and intracellular cytokine stain (ICS) assays. Functional avidity of epitope-specific T cells for some of the variants was, however, markedly reduced. These mutations which reduced avidity also abrogated recognition by epitope-specific CD8(+) T cells in a viral suppression assay. Our results indicate that "cross-reactive" CD8(+) T-cell responses identified in ELISPOT and ICS assays using a single high concentration of variant peptide often fail to predict the recognition of cells infected with variant viruses.