Dendritic cells are human leucocyte antigen (HLA)-DR positive accessory cells, that play a critical role in the development of cell-mediated immune reactions. Since the pleural space is frequently involved in cell-mediated immune reactions, we sought to isolate dendritic cells from pleural fluid. Pleural effusion mononuclear cells (PEMCs) were obtained by Ficoll centrifugation of exudative pleural effusions recovered from 19 patients with malignant pleurisy. After double-step adherence, firmly-adherent mononuclear cells (FAMs) and loosely-adherent mononuclear cells (LAMs) were recovered. The latter cells were centrifuged on a bovine plasma albumin gradient to obtain a loosely-adherent low density fraction. Nonadherent cells were rosetted with sheep red blood cells (SRBC) to obtain a nonadherent nonrosetting (NANR) cell fraction. Mitomycin C-treated PEMCs, NANRs, FAMs, and LAMs served as stimulatory cells in mixed leucocyte reaction experiments. 3H-thymidine incorporation by purified normal allogeneic blood T-lymphocytes was assessed as an index of accessory cell function. The phenotype of NANR, FAM and LAM cells was characterized using single and double stainings with a panel of monoclonal antibodies (MoAbs). Accessory cell (AC) activity (counts per minute (cpm) x 10(3)(+/-SE); 2.5 x 10(4) AC x well(-1) of LAM (148+/-24) and NANR (108.4+/-11.2) was greater than that observed for FAM (59.3+/-9.4) and for unfractioned PEMC (13.8+/-4.9). The FAM fraction was virtually entirely composed of CD68+ HLA-DR+ mononuclear phagocytes. NANR and LAM contained 51+/-12% and 65+/-6% HLA-DR+ cells, respectively, and most HLA-DR positive cells were negative for CD3, CD19, and CD68, markers for T-, B-lymphocytes and mononuclear phagocytes. Moreover, both NANR and LAM fractions contained significant numbers of cells bearing the RFD1 surface marker, expressed on dendritic cells. These results suggest that dendritic cells are present in exudative pleural effusions, and that they may be involved in the development of cell-mediated immune reactions in the pleural space.