We used fluorescence in situ hybridization (FISH) with DNA probes derived from bivariate fluorescence activated flow sorting of primate chromosomes. In cases where human and primate karyotypes differ by chromosome rearrangements, reverse painting of primate probes resulted in a subregional delineation of the human homologous chromosomes. Probes were used from two gibbon species (Hylobates concolor and H. syndactylus) which both showed highly rearranged karyotypes. Hybridization of human chromosomes with painting probes derived from both gibbons showed that, with the exception of human chromosomes 15, 18, 21, 22 and the sex chromosomes, each chromosome was differentiated in at least two and up to six segments. These probes have been used in the analysis of various cases of constitutional chromosomal rearrangements in human pathology including complex intrachromosomal rearrangements. They were also used in a multi colour format (colour segmenting) to differentiate the entire human karyotype into 81 homologous coloured segments with probes derived from H. concolor, and 74 segments with probes derived from H. syndactylus. The addition of colours not only simplifies chromosome identification compared to the analysis of classical banding based on grey values, but colour segmenting also provides simple coloured landmarks for further fine analysis by classical banding.