All the chromosomes from isogenic TOP1 and top1 strains have similar mobility on pulsed-field gels except for chromosome XII, which fails to migrate into the gels in top1 mutants. Chromosome XII contains the tandem repeats of rRNA-encoding DNA (rDNA). When a segment of chromosome XII containing only rDNA is transferred to chromosome III by a recombination event, chromosome III fails to enter a pulsed-field gel in extracts from top1 strains, indicating that the aberrant migration of chromosome XII in top1 mutants is caused by the presence of rDNA. Failure of chromosome XII to migrate into a pulsed-field gel occurs only in preparations from exponentially growing top1 cultures and not in preparations from stationary-phase top1 cultures. rDNA from a top1 strain does enter the gel if it is cut with an enzyme (Pst I) that cuts the tandem rDNA array into single 9-kb repeat units, indicating that more than a single repeat unit is required to maintain the aberrant structure.