Rare individuals of Xenopus laevis exhibit frequent initiation of transcription in the spacers of oocyte ribosomal DNA (rDNA). Using electron microscopy we have characterized spacer transcription in such an individual and have confirmed that the sites of transcription initiation correspond to the imperfectly duplicated promoters ("Bam islands") present in the X. laevis rDNA spacer. We have cloned a repeat unit containing a gene and a spacer from this individual and have injected the recombinant plasmid, pXlr 164, into oocytes of other X. laevis individuals. In electron microscope preparations the spacers of some of the cloned repeats were transcribed by RNA polymerase I. This demonstrates that the ability to initiate transcription at the Bam islands is a property of the spacer DNA. On pXlr 164, initiation in the spacer occurred about 5% as frequently as transcription from the gene promoter. However, transcribed spacers were as closely packed with RNA polymerase as was the gene. We conclude that polymerase I promoters may vary over a wide range in the frequency with which they "activate" but that once activated all can load polymerases to maximal density. The presence or absence of spacer transcription had no observable effect on either the frequency of activation or the density of polymerase loading of the gene immediately downstream. A subclone, pXlr 264, containing only spacer DNA also showed regular initiation and termination, providing further evidence that there is an effective "fail-safe" termination signal 225 base pairs upstream from the rRNA gene initiation site.