Mouse and human 5 S rRNA genes which are, respectively, active and inactive in a human cell-derived transcription system have been characterized. Both contain several base pair substitutions, relative to the gene encoding the prominent 5 S rRNA, indicating that they are variant genes. Both the mouse gene and a Xenopus 5 S rRNA gene are transcribed in systems reconstituted either with the Xenopus 5 S gene-specific factor (IIIA) and other factors from human cells or with the human IIIA equivalent and other factors from amphibian cells. This suggests that structural aspects of the factors relevant to their interactions with each other and with DNA have been conserved and that transcription of mammalian genes is mechanistically similar to that of amphibian genes which employ an internal control region (the site of interaction of IIIA). The latter is also indicated by the demonstrated interaction of the purified Xenopus IIIA factor with both the active mouse and the inactive human gene at intragenic sites corresponding to the internal control site of the amphibian gene; this is observed despite the presence of sequence variations in this region in both of the mammalian genes. Thus, the complete inactivity of the human gene and the lowered activity of the murine gene (relative to the amphibian gene) is not necessarily due to a failure to bind the 5 S gene-specific factor. It is suggested that some control region sequences may be directly or indirectly involved in other factor interactions and the possible roles of specific bases within this region in productive transcription complex formation is discussed. The sequence analyses also suggest alternate termination sequences for class III genes.