We have used a mouse cell transformant generated by human chromosome-mediated gene transfer (CMGT) to explore the use of cell surface antigens in the identification of fragments of human chromosomes retained by somatic cell hybrids. The transformed line, 21-30b, contained an intact rear-ranged human chromosome, and could be shown by isozyme analysis to contain genetic material from chromosomes 9 and X. By using the transformant as an immunogen in mice, it was also possible to produce antiserum to human-specific surface antigens. Using genetically characterized human X rodent hybrid lines, the genes controlling expression of these antigens could be localized to 11per----11p13, segregating concordantly with surface antigen S3. These conclusions were possible despite the fact that the presence of chromosome 11 in the transformant was not detectable by the presence of chromosome specific isozyme LDH-A or surface antigens W6/34 and 4F2. Finally, the fluorescence-activated cell sorter (FACS) was used to fractionate the transformant cells into antigen positive and negative subpopulations. This resulted in the isolation and characterization of four additional chromosome rearrangements involving interspecies chromosome translocations. This work demonstrates the value of chromosome-specific surface antigens and the FACS in the evaluation of human chromosome fragments retained by interspecies hybrids.