The AsiA protein, encoded by bacteriophage T4, inhibits Esigma70-dependent transcription at bacterial and early-phage promoters. We demonstrate that the inhibitory action of AsiA involves interference with the recognition of the -35 consensus promoter sequence by host RNA polymerase. In vitro experiments were performed with a C-terminally labelled sigma factor that is competent for functional holoenzyme reconstitution. By protease and hydroxyl radical protein footprinting, we show that AsiA binds region 4.2 of sigma70, which recognizes the -35 sequence. Direct interference with the recognition of the promoter at this locus is supported by two parallel experiments. The stationary-phase sigma factor containing holoenzyme, which can initiate transcription at promoters devoid of a -35 region, is insensitive to AsiA inhibition. The recognition of a galP1 promoter by Esigma70 is not affected by the presence of AsiA. Therefore, we conclude that AsiA inhibits transcription from Escherichia coli and T4 early promoters by counteracting the recognition of region 4.2 of sigma70 with the -35 hexamer.