The genes encoding the precursor of 18S, 5.8S and 25S ribosomal RNAs are transcribed in the nucleolus by RNA polymerase I. Unlike rRNA gene promoters in animals which differ substantially across species boundaries, plant rRNA gene promoters share sequence similarity for several nucleotides upstream and downstream of the transcription start site (+ 1). The conserved sequence consists of a near-consensus TATA box, a critical promoter element of most genes transcribed by RNA polymerase II and certain genes transcribed by RNA polymerase III, followed by a cluster of four to six guanosines. Using transient expression of cloned promoter deletions in Arabidopsis thaliana protoplasts, it is shown that the 5' boundary of the Arabidopsis rRNA gene promoter is located between -55 and -33 and the 3' promoter boundary is approximately +6. The critical role of the TATA element at the initiation site was demonstrated by altering this region using tandem point mutations within constructs containing essentially complete intergenic spacer sequences from -2590 to +6. The initiation site mutations either abolished transcription or dramatically reduced transcript abundance relative to the wild-type promoter. In some mutants, transcription initiation was shifted to a new site, suggesting a role for the TATA-like initiator region in both start site selection and promoter strength. It is suggested that minimal rRNA gene promoters might be similar in design to minimal promoters of protein-encoding genes, many of which utilize an initiator element, or INR, as an important promoter element located directly at the transcription start site.