We have studied the initiation of human ribosomal RNA synthesis in vitro using a cell-free polymerase I transcription system derived from HeLa cells and cloned human ribosomal DNA containing the site of initiation for ribosomal RNA synthesis. Mapping of the RNA products by run-off assays and high-resolution S1 nuclease analysis indicated that transcription in vitro initiates at a unique site and that the RNA has the same 5' terminus as in vivo precursor ribosomal RNA isolated from nuclei of HeLa cells. To provide additional evidence for the initiation site of ribosomal RNA transcription, dinucleoside monophosphates complementary to a sequence on the template have been used as primers for ribosomal RNA synthesis. When the concentrations of the four nucleoside triphosphates in the transcription reaction were reduced to 10 microM, [alpha-32 P]GTP was no longer incorporated into the run-off transcript. Under these conditions, we then tested a variety of dinucleotides for their ability to initiate promoter-specific RNA synthesis, and found that GpC exhibited maximum stimulation. We have also determined that the human cell-free system exhibits a significant degree of template specificity and is able to transcribe ribosomal DNA derived from human and rhesus monkey cells but not from mouse cells.