A method combining flow sorting and molecular cytogenetic techniques for the identification of unknown marker chromosomes is described. In this study, the bladder tumor cell line J82 was used, which was known to carry a marker chromosome of the size of chromosome 7 in every cell. From the cytogenetic analysis of Q-banded metaphase cells, it was shown to be composed of approximately 40% presumably the greater part of chromosome 20 and for the rest microscopically unidentifiable material. This marker chromosome was found using flow cytometric analysis to form an independent peak and hence was suitable for isolation using dual-parameter sorting after staining with Hoechst 33258 and chromomycin A3. Subsequently, the marker was isolated by dual-parameter sorting. DNA amplification of 300 isolated chromosomes by polymerase chain reaction (PCR) using the Alu-primer Bk33 and the LINES-primer LH5 was carried out. After purification of the amplified product, a yield of 5 microns of DNA was obtained. The DNA was labelled using Bio-11-dUTP and applied to human lymphocyte metaphase cells in a suppressive in situ hybridization procedure. Fluorescence was visible over chromosome 20 and over the distal one-half of 6p. Together the fluorescent regions accounted for only approximately 60% of the marker length, indicating a possible duplication of chromosome 20 material. This was confirmed by applying bicolor in situ hybridization using chromosome 6- and 20-specific DNA libraries to metaphase cells of the J82 cells.