Genomic Xenopus borealis oocyte-specific 5S DNA (Xbo) contains clusters of 5S rRNA genes. The number of genes varies among clusters, and the distance between genes within a cluster is about 80 nucleotides. The spacer DNA between gene clusters is AT-rich and heterogeneous in length due in part to variable numbers of a tandemly repeated 21 nucleotide sequence. A cloned fragment of Xbo 5S DNA (Xbo1) containing three 5S rRNA genes has been sequenced. The sequences of Xbo1 genes 1 and 2 are very similar to the dominant 5S RNA sequence, whereas 15 of the 120 residues in the third gene are different. The sequence of gene 3 is as different from the dominant gene sequence as the X. laevis pseudogene is from the 5S RNA gene. Sequence analysis of genomic DNA shows that gene 3 is an abundant component of the multigene family. All three genes are transcribed when added to an extract of X. laevis oocyte nuclei, and a fragment of Xbo1 lacking the AT-rich spacer DNA and the 5' end of the first gene supports transcription of genes 2 and 3 in this in vitro system. Thus the 80 nucleotides preceding each 5S gene are sufficient for promoter function. Nucleic acid sequences preceding several eucaryotic genes that are transcribed by RNA polymerase III were analyzed and the following common features were found: a purine-rich region; at least one direct repeat; the absence of dyad symmetry; transcription beginning with a purine; a pyrimidine residue immediately preceding the first nucleotide of the gene; and the oligonucleotides AAAAG, AGAAG and GAC, located approximately 15, 25 and 35 nucleotides, respectively, before the start of transcription. The 10 base pair (bp) spacing between the homologous oligonucleotides is that expected for a recognition signal on one face of a DNA double helix. The extensive sequence differences between most of the spacers that precedes these genes make the three conserved oligonucleotides more striking. Parts of the 5' flanking regions of the three Xbo1 gene (-12 to -40), which include the conserved oligonucleotides, are identical. In contrast, 7 of the first 11 nucleotides that precede the third 5S RNA gene in Xbo1 differ from those that precede the first gene. The sequences following the X. borealis oocyte and somatic 5S genes are identical in 12 of the first 14 residues and contain two or more T clusters, as does the corresponding region of X. laevis oocyte 5S DNA. The 3' sequences of the Xenopus 5S rRNA genes and several other eucaryotic genes contain features in common with procaryotic transcription termination sites. The 3' end of the gene is GC-rich and contains a dyad symmetry. Termination occurs in an AT-rich region containing one or more T clusters on the noncoding strand.