Autologous, virus-transformed lymphoblastoid cell lines were established by using peripheral blood lymphocytes from rhesus monkeys that were previously immunized with recombinant human immunodeficiency virus type 1 strain IIIB glycoprotein 160. These autologous cell lines were used to present human immunodeficiency virus type 1 viral antigens in a processed and cell-associated manner to T lymphocytes. This was accomplished by either infecting the cells with recombinant vaccinia viruses or pulsing them with synthetic peptides and then subjecting them to a mild fixation step with glutaraldehyde. Fixed antigen-presenting cells were then used as stimulator cells in vitro to measure cell-mediated immune responses. Both the vaccinia virus-infected and peptide-pulsed autologous cells stimulated antigen-specific cellular proliferative responses. The magnitude of the responses correlated with the immunization histories of the animals and other measures of immunity, such as antibody titers. Autologous vaccinia virus-infected cells were also capable of inducing the in vitro maturation of CD4+ and CD8+ precursor cytotoxic T lymphocytes into antigen-specific mature cytotoxic T lymphocytes. The use of stimulator cells to present viral peptides in a cell-associated manner appeared to be a very sensitive and versatile manner in which to measure cell-mediated immune responses with peripheral blood lymphocytes from nonhuman primates. It is likely that a similar approach will function with peripheral blood lymphocytes from humans.