Regulation of nondisjunction of the rye B chromosome was investigated using the cytidine analog 5-azacytidine, which affects DNA methylation. The B chromosome of rye normally undergoes nondisjunction at first pollen grain mitosis and is stable at all other mitotic nuclear divisions. Observations on mitosis in the control root cells showed normal chromosome behaviour, whereas the treated roots were characterized by a high frequency of anaphases with lagging chromosomes showing evidence of failure of chromatid separation. The occurrence of nondisjunction was confirmed by the presence of variable numbers of B chromosomes between cells within roots, whereas the A-chromosome number remained constant. These results suggest that the epigenetic process by which somatic cells maintain the inactivity of the gene(s) responsible for B chromosome nondisjunction, between fertilization and meiosis, is mediated through DNA methylation.Key words: B chromosome, DNA methylation, meiotic reprogramming, 5-azacytidine, rye.