HIV-1 envelope-specific CD4+ T cell lines were established simultaneously from PBMC and lymph node mononuclear cells of two HIV-1-infected patients. Three recombinant envelope proteins were used to establish the CD4+ T cell lines: gp160NL4-3, gp120IIIB, and gp120MN. Six T cell lines were established from the first patient, one for each Ag from each compartment, and four T cell lines, two per compartment, were established from the second patient. Each line was challenged with a panel of overlapping peptides spanning the entire gp120 sequence to define its T cell epitope specificity. The pattern of recognition for all the lines from any given patient was similar between compartments. Each patient had a different pattern of peptide recognition. TCR analysis showed a heterogeneous usage of Vbeta between lines with same peptide specificity and established from different compartments. These data suggest that the cellular immune response does not phenotypically vary between the peripheral blood and lymph node compartments, but demonstrates genotypic heterogeneity, showing possible redundancy of the immune response to HIV-1 gp160.