Effects of mutations around the phage SP6 transcription initiation site on SP6 RNA polymerase's selection of initiation site were studied. In the in vitro transcription reactions, the limiting concentration of a ribonucleotide causes the SP6 RNA polymerase to stall long enough only at the positions of the limited nucleotide and dissociate from the elongation complex. As a result, a series of RNA oligomers comprises a sequencing ladder, and abortive initiation cycling products up to 6-mer are made in high yield. Precise sizing of the product RNAs from the elongation pausings determined the initiation site of each mutant. When the wild-type +1 G is changed to C or A without change in the upstream sequence including TATA from -4 to -1, transcription still starts only at the +1 site. But, the mutant containing TATCC from -4 to +1 C. We propose that the phage SP6 RNA polymerase selects the initiation site precisely at a certain distance from a direct contact point in the upstream promoter sequence, regardless of the species of initiating nucleotide. It is also suggested that the sequence-dependent perturbations of DNA helical structure, for example D to B form, may shift the initiation site.