The nucleotide sequence of a 4.5-kilobase (kb) segment of rye DNA (in the clone pScR4) containing the major spacer region of ribosomal DNA, 0.25 kb of the 5′ end of the 18S gene and approximately 1.2 kb of the 3′ end of the 26S gene is presented. The 5′ end of the 18S gene, and most of the 3′ end of the 26S gene, could be readily defined by comparisons with previously published sequences. The major spacer region was dominated by the presence of 10 repeating units, with a basic length of 134 base pairs (bp), which showed sequence variability in that deletions of sections of the sequence as well as base-pair changes were common. A portion of the sequence which was common to almost all the spacer repeats and is also found in the corresponding wheat spacer repeats (in pTa250), with only two mismatches (using a consensus sequence of the rye spacer repeats as a comparison), is GCGACATGGAAAACCGGGCAAAACCACGTAC. The occurrence of this conserved sequence between more divergent sequences in both wheat and rye suggests that it may be important in forming the pretranscription complex involving RNA polymerase I and other protein cofactors. Probes were isolated from the spacer region to preferentially assay the rye rDNA in wheat backgrounds. In situ hybridization studies revealed that when 1R was the only NOR-containing chromosome present the rDNA was in a dispersed state. This dispersed state correlates with nucleolus formation and presumably with rDNA transcription. In the presence of chromosomes 1B and (or) 6B of wheat (these chromosomes carry NOR loci) the dispersed state of rye rDNA was generally suppressed, but not completely, suggesting that 1R retains some potential for activity.Key words: rye, NOR R1 locus, nucleotide sequence, transcription.