Peritoneal exudate lymphocytes (PEL) from immune guinea pigs that adhere to antigen-pulsed macrophages (MØ) were cultured for 1 week to yield a population enriched in antigen-specific (selected) T cells. These cells bind specifically within hours to fresh autologous antigen-pulsed MØ. The dissociation of these selected PEL from antigen-pulsed MØ was studied. No evidence was obtained that factors in the culture medium play a role in dissociation. Lymphocytes that have dissociated from antigen-pulsed MØ are usually fully capable of rebinding to MØ freshly pulsed with antigen, suggesting that there is no deficiency in the lymphocytes ability to bind. In contrast, readding antigen to cultures during incubation prevents the predicted dissociation. Moreover, repulsing MØ cultured without selected PEL restores their capacity to bind fresh selected PEL. These findings indicate that decay of antigen associated with MØ is the major mechanism underlying the observed dissociation.