We present evidence for intrathymic presentation of soluble circulating antigens in vivo. Our results show that proteins of different molecular weight enter the mouse thymus rapidly after i.v. injection. The intrathymic presence of antigen was assayed by proliferation of cloned antigen-specific T helper cells, which were cocultured with purified thymic stromal cells; stromal cells were isolated and purified as lymphostromal cell complexes, which preexist in vivo. Antigen presentation copurified with non-adherent medullary dendritic cells (DC) (interdigitating cells). I-A- cortical macrophages forming thymocyte rosettes in vivo and I-A+ cortical epithelial cells forming thymic nurse cells (TNC) in vivo did not act as antigen presenting cells (APC) after antigen pulsing in vivo or in vitro. Thymic APC turn over physiologically and are rapidly replaced (within 2-5 wk) after lethal irradiation by donor bone marrow-derived cells. The frequency of thymocyte-DC interactions in vivo strictly correlates with thymic T cell differentiation, and is independent of the immune status of the animal. Fetal thymic APC seem to be secluded from antigen in the maternal circulation. Thymic DC-ROS probably represent the microenvironment where maturing T cells first encounter non-MHC antigens in the context of self-MHC antigens.