The tandemly repeated human ribosomal RNA (rRNA) genes contain a region of size heterogeneity that is present in the nontranscribed spacer of every individual examined. This heterogeneity has been previously examined by Southern analysis of BamHI-digested human DNA. Using a ribosomal DNA (rDNA) probe specific for the 3' end of the 28S rRNA gene, at least four discrete sizes of BamHI fragments were seen in human populations. Molecular analysis of the cloned DNA from this region reveals tandem duplication of a segment of spacer rDNA located 388 base pairs (bp) 3' to the end of the 28S ribosomal RNA gene. Five hundred fifty bp of DNA, flanked on either side by a 150-bp repeated element, is either duplicated or deleted to produce a series of spacers that differ in size by 850 bp. These duplications/deletions appear to be the product of unequal homologous exchange, mediated by the small repeated element. Thus, human rDNA fragments cloned in lambda vectors and propagated in E. coli generate the same apparent size variation seen in genomic DNA. This study suggests that unequal homologous exchange is the molecular basis for the observed length heterogeneity in the spacer rDNA and may be a common mechanism for the generation of human genetic diversity.