We studied RNA polymerase I transcription in cells transfected with a plasmid, prHuTK, containing the herpes simplex virus tk gene fused to a human rRNA promoter. Primer extension analysis of tk RNA isolated from COS cells transfected with prHuTK reveals that transcription from the RNA polymerase I promoter is highly efficient and initiates at the same position used for the synthesis of endogenous rRNA in HeLa cells. The RNA products derived from prHuTK are distinguishable from normal RNA polymerase II transcripts of tk in that they are not polyadenylated, are extremely unstable, and are found predominantly in the nucleus. Moreover, the transcription observed is resistant to 300 micrograms of alpha-amanitin per ml. These results strongly suggest that prHuTK transcription is under the control of the human rRNA promoter and RNA polymerase I. To further characterize the activity of the human rDNA promoter in vivo, a series of 5' and 3' deletion mutants was tested in this transfection assay. The deletion analysis indicates that a core region of ca. 40 base pairs overlapping the initiation site is critical for transcription. In addition, a region between nucleotides -234 and -131 upstream from the core sequence serves to modulate the efficiency of transcription. Insertion into prHuTK of additional ribosomal nontranscribed spacer DNA or the simian virus 40 enhancer element has no apparent effect on the promoter activity. Surprisingly, RNA polymerase II transcripts synthesized at low levels from two start sites within the core control element of the wild-type RNA polymerase I promoter are activated upon deletion of upstream RNA polymerase I promoter sequences. However, these RNA polymerase II transcripts are not expressed from the endogenous rRNA promoter.