Probe DNA that binds preferentially to the centromeric region of human chromosomes 8 was synthesized. Alpha satellite probe DNA molecules were selectively amplified from sorter-purified human chromosomes 8 by in vitro DNA amplification using the polymerase chain reaction (PCR). Probe labeling was performed during PCR by incorporation of biotinylated deoxyuridine. In situ hybridization of unpurified probe DNA comprised of alpha satellite monomer and higher molecular weight DNA fragments with metaphase chromosome spreads showed binding to the centromeric regions of numerous chromosomes. However, blocking with unlabeled total human alphoid DNA dramatically reduced crosshybridization to chromosomes other than 8. Under these conditions, the degenerate probe DNA allowed unambiguous visualization of domains occupied by centromeric DNA of chromosome 8 in metaphase spreads and interphase cell nuclei, thus greatly facilitating the detection of numerical chromosome aberrations in tumor cells. In situ hybridization of size-fractionated alpha satellite DNA identified the monomeric fraction as the major cause of crosshybridization. Alpha satellite dimers and higher molecular weight DNA fragments showed relatively high specificity for human chromosomes 8.