We report the physical order of three alphoid DNA subsets on human chromosome 22 determined by a combination of low- and high-resolution cytological mapping. Multicolor fluorescence in situ hybridization was performed on metaphase chromosomes, interphase nuclei and extended chromatin preparations. The results visually demonstrate the presence of three distinct alphoid DNA domains at the centromeric region of chromosome 22. Two domains appear adjacent by extended chromatin hybridization, while the third one is separated by DNA that does not hybridize with any of our probes. Our data demonstrate the applicability of interphase mapping for ordering alpha satellite DNA repeat arrays. However, in our experiments, the relationship between the extremities of repeat arrays could only be studied by extended chromatin experiments.