Upstream of the mouse 45S pre-ribosomal RNA promoter there are regions that are involved in enhancement of pre-rRNA transcription, origin of replication and promotion of amplification. We report that in different mouse strains and cell lines the enhancer region, which overlaps with the origin of replication, is hypo-methylated. Contrary to that the amplification-promoting sequences 1 and 2, identified upstream in the intergenic spacer, are hypermethylated. Hybridization results also suggest that amplification-promoting sequence 1 is repeated in the genome.