CD58 (LFA-3), a heavily glycosylated protein of 40-70 kDa, is expressed on a broad range of hematopoietic and non-hematopoietic cells. It serves as a physiological ligand of the CD2 receptor, present on T cells and natural killer cells, and plays, thus, an important role in lymphocyte adhesion and T cell activation through CD2. Whereas several epitopes and their respective function are known for CD2, a similarly detailed characterization of CD58 is still lacking. We raised a panel of novel murine monoclonal antibodies (mAb) against recombinant human CD58 and describe here the identification of six structurally and/or functionally distinct epitopes on the CD58 molecule. All epitopes were found to be present in equal numbers on a wide range of CD58+ cells, none of them being differentially up-regulated following cell activation or malignant transformation. Two of these epitopes represent functionally relevant sites, involved in binding of CD58 to CD2 and T cell activation via CD2. One further epitope appears to be selectively involved in CD58-mediated activation, whereas the other three displayed no functional effects. The new mAb allow for the first time the detection of CD58 in enzyme-linked immunosorbent assays and immunofluorescence while bound to its receptor CD2 in human serum or on freshly isolated blood cells. Finally, one mAb was found to specifically cross-react with T11TS, the equivalent of CD58 in sheep.