Genomic in situ hybridisation was used to confirm that Nicotiana rustica (2n ¼ 4x ¼ 48) is an allotetraploid between N. paniculata (2n ¼ 2x ¼ 24, maternal P-genome donor) and N. undulata (2n ¼ 2x ¼ 24, paternal U-genome donor), their progenitors or species closely related to them. Fluorescent in situ hybridisation showed that N. paniculata has one 5S and two 18-5.8-26S rDNA loci whereas N. undulata has an additional 18-5.8-26S rDNA locus. N. rustica has the sum of the loci found in these putative parents. The sizes of the 18-5.8-26S rDNA loci indicate that the number of rDNA units on the U-genome chromosomes has amplified; perhaps this is associated with a concomitant reduction in the number of units on P-genome chromosomes. Restriction fragment length polymorphism analysis of the intergenic spacer (IGS) of the 18-5.8-26S rDNA units in N. rustica and the two progenitor diploids revealed that about 80% of IGS sequences in N. rustica are of an N. undulata type and 20% of N. paniculata type. These data indicate that interlocus sequence homogenisation has caused the replacement of many N. paniculata-type IGSs in N. rustica with an N. undulata-type of sequence. It is probable that subsequent to this replacement there has been sequence divergence at the 5 0 end of the IGS. As in tobacco, an allotetraploid between N. sylvestris and N. tomentosiformis, the direction of the IGS interlocus conversion is towards the paternal genome donor.