We have studied RNA synthesis in homogenates of nuclei manually isolated from mature oocytes of Xenopus laevis. Transcription of the endogenous template is completely resistant to alpha-amanitin and represents primarily transcription of the amplified ribosomal genes. Three lines of evidence demonstrate that RNA polymerase I is reinitiating RNA chains in this system. First, transcription continues in the homogenates for at least 8 h. During this time, 50 to 100 RNA copies are synthesized per gene. Second, inhibitors of initiation, such as heparin and sodium dodecyl sarcosinate, cause synthesis to plateau within 60 min with less than 15 RNA copies synthesized per gene. Third, incorporation of gamma-thio nucleoside triphosphates demonstrates that reinitiation is occurring. When RNA is synthesized in the presence of adenosine-5'-O-(gamma-thiotriphosphate) and guanosine-5'-O-(gamma-thiotriphosphate), 3 to 10% of the newly made RNA binds to a Hg-agarose affinity column. After 7.5 min of synthesis the gamma-thio-initiated RNA binding to Hg-agarose hybridizes exclusively to a restriction fragment contining the first 2250 base pairs of the gene. At longer times of synthesis, the gamma-thio-initiated RNA hybridizes more and more to a restriction fragment located further down the gene. The length distribution of this RNA on gels is consistent with its hybridizaton behavior and these data suggest an in vitro chain growth rate of 2 to 5 nucleotides/s. All of the gamma-thio-initiated RNA hybridizes to the H or coding strand of rDNA. The results are consistent with initiation in vitro occurring at the same site used in vivo. Addition of cloned ribosomal genes to the system causes no stimulation of transcription until it is added in a 4000-fold excess over the endogenous ribosomal genes. At that excess it is symmetrically transcribed by RNA polymerase III.