The 10 kDa bacteriophage T4 antisigma protein AsiA binds the Escherichia coli RNA polymerase promoter specificity subunit, sigma 70, with high affinity and inhibits its transcription activity. AsiA binds to sigma 70 primarily through an interaction with sigma 70 conserved region 4.2, which has also been implicated in sequence-specific recognition of the -35 consensus promoter element. Here we show that AsiA forms a stable ternary complex with core RNA polymerase (RNAP) and sigma 70 and thus does not inhibit sigma 70 activity by preventing its binding to core RNAP. We investigated the effect of AsiA on open promoter complex formation and abortive initiation at two -10/-35 type promoters and two "extended -10" promoters. Our results indicate that the binding of AsiA to sigma 70 and the interaction of sigma 70 region 4.2 with the -35 consensus promoter element of -10/-35 promoters is mutually exclusive. In contrast, AsiA has much less effect on open promoter complex formation and abortive initiation from extended -10 promoters, which lack a -35 consensus element and do not require sigma 70 conserved region 4.2. From these results we conclude that T4 AsiA inhibits E. coli RNAP sigma 70 holoenzyme transcription at -10/-35 promoters by interfering with the required interaction between sigma 70 conserved region 4.2 and the -35 consensus promoter element.