Specific detection of fibroblasts has been one of the unsolved problems in cell biology. Because monoclonal antibodies (MoAbs) might provide an easy and reproducible method of fibroblast detection, we have produced a panel of MoAbs raised against cell surface proteins of human dermal fibroblasts. Using flow cytometry and immunohistochemistry, we have shown that two of these MoAbs, FibAS01 and FibAS02, react exclusively with human fibroblasts. They do not react in vitro with human keratinocytes, endothelial cells, or blood cells. Immunohistologic experiments investigating the binding pattern of the MoAbs FibAS01 and FibAS02 in cryostat sections of different tissues confirmed the flow cytometric results. In human skin, the antibodies exclusively labeled fibroblasts. In other human tissues such as lymph nodes, placenta, kidney, muscle, thyroid gland, gall bladder, cartilage, and tendon, the specificity for fibroblasts was borne out. Neither antibody reacts with fibroblasts from mouse, rat, or pig. The isotype was defined as an IgG1 for both. By western blot analysis, both antibodies detected a molecule of 60-65 kDa under reducing and nonreducing conditions. By immunoelectron microscopy, we observed the antigens on the cell surface without any clustering at specific sites. These data demonstrate that the two MoAbs, FibAS01 and FibAS02, exclusively recognize human fibroblasts.