Barley nucleolus organizing regions (NORs) were previously found to behave as prominent aberration hot-spots after treatment with some restriction endonucleases. The ability of MspI for directed induction of double-strand breaks in barley ribosomal DNA was further analyzed. Ionizing radiation-produced strand breakage within the ribosomal gene clusters was also a subject of investigation. Reconstructed barley karyotypes T1586 and T35 with normal and increased expression of rRNA genes were utilized to evaluate the relationship between transcriptional activity and damage induction. Scanning densitometry of the hybridization profiles revealed that MspI is generating double-strand breaks in barley rDNA with efficiency being independent from the NOR activity. Damage induction observed after treatment with gamma-rays was also not influenced by the transcriptional status of the ribosomal genes. A tendency towards restoration of rDNA integrity after irradiation of both germinating and dry seeds was observed which is indicative for the efficient recovery of double-strand breaks in barley ribosomal DNA.