A characteristic karyotypic feature of mouse chromosomes is the presence of large blocks of heterochromatin in the vicinity of the centromeres. Breakage inside this centromeric heterochromatin might result in the formation of abnormal chromosomes, very similar to the metacentric chromosomes derived from Robertsonian fusion. X-rays are very efficient in inducing these Robertsonian fusion-like configurations (RLC) in cultured mouse splenocytes. Observed frequencies of these RLC increase in a linear-quadratic manner with dose. Two types of RLC were found. The first type (approximately 70% of induced RLC) has heterochromatin only in the middle of the chromosome and appears as a metacentric chromosome, whereas the other type (approximately 30%) has a heterochromatic block inside the chromosome arm (and has the appearance of a dicentric chromosome). Induced RLC are difficult to classify as either a stable or unstable aberration, based only on traditional cytogenetic techniques such as C-banding. Here we describe a cytogenetic approach to achieving better insight into the molecular organization of RLC. We utilized two-colour fluorescence in situ hybridization (FISH) using a combination of mouse minor satellite DNA probe and telomeric probe. Over 90% of RLC did not have detectable minor satellite arrays inside the interstitial heterochromatin. Consequently, most of the RLC of the first type should be classified as acentric fragments and those of the second type as translocations. The chance of inducing true Robertsonian fusions in mouse splenocytes by X-rays is < 2.5% based on the total RLC observed.