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A length difference of about 50 bp in the EcoRI fragment B of the rDNA from two different strains of Saccharomyces cerevisiae has been mapped in detail by sequencing of cloned fragments. This 2.4 kb EcoRI fragment contains the start of the 35S rRNA gene at one end and the 5S rRNA gene in the middle flanked by non-transcribed spacers, NTS1 and NTS2. The difference appeared as short deletions or insertions in five regularly spaced regions within the 1 kb NTS1, 3' to the 5S rRNA gene. The same regions of heterogeneities were displayed when all available sequence data of the NTS1 were compared. Four of the variable regions are located 160-170 bp apart, indicating that they might represent linker sequences between phased nucleosomes. Two variant clones, differing in the length of one subfragment of NTS1, were isolated for each strain. In both cases these represented the major variants among chromosomal NTS1 as revealed by sequencing of genomic fragments.