The T7, T3, and SP6 RNA polymerases represent a highly homologous family of enzymes that recognize similarly homologous promoter DNA sequences. Despite these similarities, the enzymes are highly specific for their respective promoters. Studies of mutant RNA polymerases have linked a specific amino acid residue in the protein to recognition of bases at positions -11 and -10 in the promoter [Raskin, C. A., et al. (1992) J. Mol. Biol. 228, 506-515]. In kinetic analyses of transcription from synthetic promoters containing base-analog substitutions, we have recently shown that at positions -11 and -10 of the T3 promoter, T3 RNA polymerase recognizes functional groups along the nontemplate strand wall of the major groove [Schick, C., & Martin, C. T. (1993) Biochemistry 32, 4275-4780]. We now extend these studies to the homologous region of the T7 promoter. The results confirm extrapolations from the T3 system and show that T7 RNA polymerase recognizes corresponding functional groups at positions -11 and -10 of the T7 promoter. The results are consistent with a direct readout model for recognition of these bases [Raskin, C. A., et al. (1992) J. Mol. Biol., 228, 506-515], in which the 6-carbonyl and 7-imino groups of the nontemplate guanine at position -11 and the 6-amino group of the nontemplate adenine at position -10 of the T7 promoter are directly involved in binding. The results further support an overall model for promoter recognition in which the enzyme binds to one face of the duplex DNA in this upstream region of the promoter.