The acrocentric chromosomes of 18 unrelated individuals were analyzed by sequential staining by the chromomycin A3/methyl green R-banding technique to identify the chromosomes, followed by an indirect immunoperoxidase technique to detect 5-methylcytosine (5MeC)-rich DNA. The short arms of both chromosomes 15 usually (92% of the chromosomes) had a large collection of 5MeC-rich DNA, which was always rich in AT base pairs. Much less commonly (11% of the possible occasions), a collection of 5MeC-rich DNA was seen on the short arm of a chromosome 13, 14, 21 or 22, and this DNA was always rich in GC base pairs. Sequential distamycin A/DAPI (DA/DAPI) and R-banding studies were carried out in 13 of these 18 individuals. There was bright DA/DAPI fluorescence of the 5MeC-rich region on the short arm of chromosome 15 but not on that of any other acrocentric chromosome. One implication of these findings is that bisatellited or other abnormal chromosomes that are DA/DAPI negative and 5MeC positive cannot be derived from number 15. In the case of a de novo chromosome of this type, the specific origin from any other acrocentric chromosome could be demonstrated by examining 5MeC-binding of the parental chromosomes.