The intergenic spacer region of the Xenopus laevis ribosomal DNA contains multiple elements which are either 60 or 81 base pairs long. Clusters of these elements have previously been shown to act as position- and distance-independent enhancers on an RNA polymerase I promoter when located in cis. By a combination of deletion and linker scanner mutagenesis we show that the sequences essential for enhancer function are located within a 56-base-pair region that is present in both the 60- and 81-base-pair repeats. Within the 56-base-pair region one linker scanner mutation was found to be relatively neutral, suggesting that each enhancer element may be composed of two smaller domains. Each 56-base-pair region appears to be an independent enhancer with multiple enhancers being additive in effect. We review the current evidence concerning the mechanism of action of these enhancers.