Marker chromosome are small supernumerary chromosomes that are sometimes associated with developmental abnormalities. Hence, the genes involved in such cases provide an interesting approach to understanding developmental abnormalities in man. As a first step towards isolating such sequences, marker chromosomes need complete characterization. By combining chromosome isolation by flow sorting and the "degenerate oligonucleotide primed - polymerase chain reaction", we have constructed a DNA library specific for a marker chromosome found in a child with severe developmental abnormalities. We used fluorescent in situ hybridization of the library onto normal metaphase spreads ("reverse chromosome painting") and were thus able to determine that the marker consists of the centromeric part of chromosome 7, the telomeric region of the long arm of chromosome 5 and the telomeric region of the short arm of the X-chromosome. Subsequently, we hybridized normal chromosome-specific libraries of the relevant chromosomes onto metaphases containing the marker chromosome ("forward chromosome painting") and could in this manner establish the precise location of the different chromosome regions on the marker chromosome itself. This is a general approach suitable for outlining marker chromosomes in detail, and will aid the identification of the genes involved.