We have constructed hybrid plasmids bearing both Xenopus 5S RNA genes and satellite I sequences in order to test the effect of satellite DNA on 5S gene transcription. Satellite sequences inactivate 5S transcription in both HeLa S100 and Xenopus oocyte microinjection transcription assays. Inactivation of 5S transcription by satellite DNA is observed both in cis and in trans. Transcription of a tRNA gene is also precluded by satellite I DNA. The Xenopus satellite I repeat contains an RNA polymerase III transcription unit which is highly active in both assay systems. This promoter element is 10- to 25-fold more efficient than the 5S gene in transcription competition assays. This quantitative difference in affinity for transcription components may explain the inactivation of 5S transcription by satellite sequences. The satellite I promoter forms stable transcription complexes in vitro which do not dissociate for at least 30 rounds of transcription. Although stable complex formation on the satellite promoter is largely temperature independent over the range of 0-20 degrees, complex formation on both 5S and tRNA genes exhibits a steep temperature dependence characteristic of DNA helix unwinding. The DNA sequence requirements for stable complex formation on 5S genes have been determined using 5' deletion mutants.