Here, we report data on the binding of wheat nuclear proteins (NP) to the promoter region of the rDNA intergenic spacer (IGS), with emphasis on the possible effects of methylation with M-HpaII on this binding. We have found that a number of NP specifically bind to the rDNA promoter, and to upstream and downstream IGS sequences. A 240-kDa NP binds specifically to a 174-bp fragment located downstream from the transcription start point (tsp), and this fragment competes with a 135-bp subrepeat element fragment for specific binding to this 240-kDa NP. Specific binding of the 240-kDa NP to the 174-bp fragment is greatly inhibited by methylation of internal cytosine residues in its two CCGG sites, whereas NP binding to the 135-bp subrepeat element is not affected by M-HpaII methylation. A 202-bp fragment containing the tsp is partially homologous to the 174-bp downstream region fragment. Nevertheless, it neither binds the 240-kDa NP nor is its binding to NP affected by methylation of CCGG sites. Thus, multiple specific NP may be involved in rDNA transcription initiation. Methylation-dependent inhibition of rDNA transcription initiation appears to be mediated by direct interference with the binding of some of these transcription factors to their respective regulatory elements.