This paper describes the effects of 5-azacytidine on the condensation state of rye (Secale cereale L.) chromatin introduced into the wheat genome (Triticum aestivum L. cv. Beaver). The wheat cultivar Beaver carries a translocation between the short arm of rye chromosome 1R (1RS) and the long arm of wheat chromosome 1B (1BL/1RS). 1RS can be detected using genomic in situ hybridisation and carries a ribosomal DNA (rDNA) locus that can be simultaneously detected using multiple labelling strategies. The rDNA locus divides 1RS into a distal region that is gene rich and a proximal region that is gene poor and highly methylated. 1RS also carries a large block of subtelomeric heterochromatin. The drug, which acts to inhibit DNA methylation in plants, has three pronounced effects on interphase nuclei. (1) It induces aberrant condensation of the rye subtelomeric heterochromatin and in many cases induces sister chromatid separation in the subtelomeric heterochromatin of G2 nuclei. (2) Nuclei trisomic for 1RS are observed at low frequency in treated material and are probably a consequence of aberrant sister chromatid separation or condensation. (3) The drug alters normal condensation of 1RS euchromatin. However, contrary to expectation the effect is not simply to induce decondensation. The proximal region of the arm actually condenses at low levels of drug administration while the distal region remains unaltered or increases its decondensation state. Increasing the concentration of 5-azacytidine induces a biphasic response and at the highest concentration used all regions of the arm show signs of decondensation. Thus the influence of the drug on chromatin condensation depends on the genomic structure.