It is demonstrated here that a DNA segment can be sequenced by transcription reactions of the bacteriophage RNA polymerases. Addition of 3``-deoxynucleoside 5``-triphosphate (3``-dNTP) chain terminators in the standard transcription reactions produced base-specific DNA-sequencing ladders of RNA transcripts of 400 or more bases. The conditions for the T7 RNA polymerase transcription were optimized for higher yields of RNA ladders (especially those shorter than 100-mer), and for uniformity of the long and short RNA ladder bands. It was evident that optimum 3``-dATP concentration was different for different sizes of RNA. For example, the rNTPs/dNTPs ratio was optimal at 0.6:1 for RNA ladders shorter than 27-mer, at 1:1 for 27 to 37-mer, at 1.5:1 for 37 to 70-mer, at 3:1 for 70 to 80-mer and at 6:1 for longer than 80-mer. The optimum magnesium ion concentration appeared to depend not only on RNA size but also on the 3``-dATP concentration. Inclusion of inorganic pyrophosphatase appeared to reduce the non-specific band intensity, and enhance the band intensities of homo-oligonucleotide regions. The higher concentrations of enzymes were, the more deoxy-ladder bands were produced. The ladder RNAs were produced more from the short linear template by 1.5- to 2-fold than from the circular template. Not only the T7 and SP6 RNA polymerases but also three mutants and nick forms that had enhanced termination efficiency were tested. Among those, the nicked forms of both T7 and SP6 RNA polymerases produced the ladders most. Also, the nicked enzymes showed decreased tendency to produce aberrant transcription products from DNA templates having protruding 3``ends. Thus, the RNA transcript ladders could be analyzed for DNA sequencing by gel electrophoresis and mass spectrometry.