The transcriptional activity of spacer sequences flanking the rat 45S ribosomal DNA (rDNA) gene were studied. Nascent RNA labeled in in vitro nuclear run-on reactions hybridized with both 5' and 3' spacer regions. The highest level of hybridization was seen with an rDNA fragment containing tandem repeats of a 130-base-pair sequence upstream of the 45S rRNA initiation site. Synthesis of RNA transcripts homologous to this internally repetitious spacer region was insensitive to high levels of alpha-amanitin, suggesting that it is mediated by RNA polymerase I. Analysis of steady-state RNA showed that these transcripts were present at extremely low levels in vivo relative to precursor rRNA transcripts. In contrast, precursor and spacer run-on RNAs were synthesized at similar levels. This suggests that spacer transcripts are highly unstable in vivo; therefore, it may be the process of transcription rather than the presence of spacer transcripts that is functionally important. Transcription in this upstream rDNA region may be involved in regulation of 45S rRNA synthesis in rodents, as has been suggested previously for frog rRNA. In addition, the presence of transcriptional activity in other regions of the spacer suggests that some polymerase I molecules may transcribe through the spacer from one 45S gene to the next on rodent rDNA.