Publisher Summary This chapter discusses the organization and transcription of the rRNA genes of eukaryotes with an emphasis on the nucleotide sequences in relation to function. The rRNA genes (rDNAs) are organized in repeating pre-rRNA transcription units separated by nontranscribed spacers (NTS). The pre-rRNAs transcription unit consists of coding sequences corresponding to mature 18-S, 28-SY, and 5.8-S rRNA, or their equivalents, and both external and internal transcribed spacer (ETS and ITS) sequences, which are transcribed as parts of the pre-rRNA molecule, but are lost during maturation. One transcription unit and the adjoining NTS constitute one rDNA repeating unit. The tandem head-to-tail arrangement of the repeated units is the basic feature in the organization of rRNA genes in most eukaryotes. The determination of nucleotide sequences at the transcription initiation and termination sites and at the gene-spacer boundaries has revealed some general features regarding sequence conservation and divergence in rDNAs. In general, the structural and functional domains of rRNA have been conserved in evolution in spite of wide divergence in the transcribed and nontranscribed spacers.