Xenopus oocytes contain an abundant protein that binds specifically to the center of 5S RNA genes and directs their transcription by RNA polymerase III. This protein also binds to 5S RNA. We show here that transcription of cloned 5S RNA genes in extracts derived from Xenopus tissue culture cells is dependent on the intragenic control region and is inhibited by 5S RNA and by antibodies raised against the previously characterized oocyte transcription factor. Somatic cells contain a protein that is similar to the oocyte factor in charge, affinity for heparin-agarose, and antigenicity but has an apparent molecular mass about 2000 daltons greater than that of the oocyte protein. Our experiments strongly suggest that this larger protein is the transcription factor for 5S RNA genes in somatic cells. The 5S RNA may regulate its own synthesis in somatic cells by binding to this protein, which is present at a low concentration. The presence of two different proteins responsible for 5S RNA synthesis in oocytes and in somatic cells cannot by itself explain the developmental control of oocyte and somatic 5S RNA genes, because somatic cell extracts transcribe both types of gene.