SummaryWe investigated six Robertsonian translocations, including two cases of rob(13q14q), one of rob(14q21q), one of rob(13q22q), and two of rob(21q21q), by means of fluorescence in situ hybridization (FISH) using five repetitive DNA probes: two alpha-satellite DNAs (D21Z1/D13Z1 and D14Z1/D22Z1), satellite III DNA, beta-satellite DNA, and ribosomal DNA. Single color FISH successfully defined the breakpoints in four cases of the six. Since the remaining two cases, rob(13q22q) and rob(21q21q), revealed to retain rDNA, we tried to define the breakpoints in detail by dual color FISH in these rare types. In the rob(13q22q) the chromosomal breakage on chromosome 22 was likely to have occurred within the rDNA region and that the chromosome 13 breakpoint was within the alpha-satellite region. In one rob(21q21q) case we defined the breakpoint on one chromosome distal to, or within, the beta-satellite region distal to the rDNA, and the other chromosome breakage had occurred within alpha-satellite DNA. Our results underscored the power of dual-color FISH for defining the precise locations of breakpoints in Robertsonian translocations.