Bone marrow plasma cells constitute the bulk of malignant cells in multiple myeloma patients. B-lymphocytes having immunoglobulin heavy chain gene rearrangements identical to those of the malignant clone (clonally related B-lymphocytes) may function as malignant plasma cell precursors. We and others proposed the use of anti-idiotypic antibodies to isolate and study clonally related B-lymphocytes. This strategy failed until now because anti-idiotypic antibodies raised by conventional hybridoma techniques proved to react frequently with epitopes shared by different idiotypes. Recently, we succeeded in selecting specific single chain Fv antibodies from phage libraries. To select single chain Fv bearing phages specifically directed against the immunoglobulin idiotype expressed by myeloma tumor cells we panned a semisynthetic phage library against purified myeloma paraprotein Fab fragments. The selection was performed in the presence of soluble polyclonal immunoglobulin as a competitor. Three independent selections for three myeloma patients yielded 10-26 clones. Between two and seven of the selected clones were reactive with patient Fab and not with polyclonal immunoglobulin in enzyme-linked immunosorbent assays. Five out of six anti-idiotypic single chain Fvs were able to specifically stain fixed monoclonal plasma cells in myeloma bone marrow. Idiotype specificity of these single chain Fvs was confirmed by flow cytometry since they did not react with monoclonal plasma cells of other patients, a panel of nine myeloma cell lines, isolated polyclonal bone marrow plasma cells and cultured B-lymphocytes. Using these anti-idiotypic reagents we were able to detect 25 myeloma plasma cells in a background of 50000 immunoglobulin isotype-matched cells of the myeloma cell line UM-1 or 50000 donor bone marrow cells (sensitivity 0.05%). This paper shows that highly specific anti-idiotypic single chain Fv antibody fragments selected from a phage display library can be used to detect rare idiotypic cells in patient samples.