We describe a chromosome-band-specific painting method that involves (1) microdissection of the chromosome, chromosomal region or band, (2) amplification of a variety of chromosome/region/band-specific DNA fragments with the polymerase chain reaction (PCR), and (3) chromosome in situ suppression hybridization (CISS) with the direct use of the PCR products as a probe pool. With this method, it was possible 1) to paint an entire X or Y chromosome, a distal one-fourth of 2q, and only a band at 8q24.1, 2) to identify the origin of a minute marker chromosome in a mentally retarded patient, 3) to detect an X;Y translocation in another patient, and 4) to identify one human chromosome 2 in a human-mouse hybrid cell line. This method allows us to identify not only structural chromosome abnormalities at the band level, but also the origin of cytogenetically unidentifiable marker chromosomes. It will also be useful in studies of evolutionary cytogenetics.