Genes coding for rRNA 1 were among the first genes isolated in pure form, initially using the properties of actinomycin-D/CsCl gradients to distinguish between different DNA sequences, 2 and later by cloning in plasmids such as pMB9, pSC101, and pACY184, 3 and bacteriophages such as Charon 4, λgts. 4 In most organisms the rRNA genes are present as multiple copies, ranging from 5 to 10 in bacteria to several thousand in eukaryotes. 5 In eukaryotes the rRNA genes are tandemly arranged at specific chromosomal locations. 5 The repeated nature of these genes in addition to their restricted distribution in the genome has allowed their analysis in a wide range of organisms. As a result this gene unit is unique in the molecular detail available. Data from such a diverse group of organisms provide a good opportunity for assessing the changes which occur in a gene system during the course of evolution. In this review we examine recent advances in the analysis of the rDNA locus, with a particular emphasis on the spacer region, and how these studies are modifying our views on the evolution of such repeated genes.