The response of thymocytes to lectin is a standard tissue culture model for identifying cytokines such as IL-1 that are required for thymocyte mitogenesis. To study accessory cell requirements for these responses, it was necessary to deplete endogenous accessory cells with two techniques: anti-Ia and complement, and passage over nylon wool. Proliferation to Con A was then restored with 0.1-0.3% exogenous splenic dendritic cells, or 30-fold higher levels of peritoneal macrophages. The "costimulatory" action of IL-1, whereby responses to lectin were enhanced 3-10-fold, required the presence of dendritic cells. This effect of IL-1 could be reproduced by culturing the dendritic cells for 12 h in 1 U/ml human or murine rIL-1 alpha before addition to the thymocyte proliferation assay. The function of IL-1-treated dendritic cells was not blocked by a neutralizing anti-IL-1 antibody. The endogenous population of thymic accessory cells was partially characterized. A trace (0.1-0.3%) fraction of Ia+, Ig-, plastic nonadherent dendritic cells was visualized and enriched to a level of 1-10% by depleting CD4+,CD8+, and Ig+ lymphocytes. When this double-negative population was cultured with IL-1 and washed, the treated thymic dendritic cells were 10-fold more active as accessory cells. When the CD4-,CD8-, Ig- populations were depleted of dendritic cells with anti-Ia and complement, the subsequent addition of IL-1 had a second effect. Ia+ dendritic cells redeveloped over a 2-d interval, and they exhibited the same properties as resident dendritic cells in thymus and spleen. The majority were lysed by 33D1 anti-dendritic cell mAb and complement, lacked Fc receptors, and acted as powerful stimulators of the MLR and Con A mitogenesis. The development of dendritic cells did not occur with IL-2, -3, -4 or granulocyte/macrophage colony-stimulating factor or in nylon-nonadherent populations. The IL-1-dependent, Ia- precursor was not detectable in bone marrow. These results begin to analyze the endogenous accessory function of the thymus in culture. Dendritic cells actively stimulate thymocyte mitogenesis. The mitogenic action of IL-1 involves effects on resident Ia+ dendritic cells as well as a new population of thymic, Ia- precursors.