Anti-sigma70 factors interact with sigma70 proteins, the specificity subunits of prokaryotic RNA polymerase. The bacteriophage T4 anti-sigma70 protein, AsiA, binds tightly to regions 4.1 and 4.2 of the sigma70 subunit of Escherichia coli RNA polymerase and inhibits transcription from sigma70 promoters that require recognition of the canonical sigma70 -35 DNA sequence. In the presence of the T4 transcription activator MotA, AsiA also functions as a co-activator of transcription from T4 middle promoters, which retain the canonical sigma70 -10 consensus sequence but have a MotA box sequence centered at -30 rather than the sigma70 -35 sequence. The E.coli anti-sigma70 protein Rsd also interacts with region 4.2 of sigma70 and inhibits transcription from sigma70 promoters. Our sequence comparisons of T4 AsiA with Rsd, with the predicted AsiA orthologs of the T4-type phages RB69, 44RR, KVP40, and Aeh1, and with AlgQ, a regulator of alginate production in Pseudomonas aeruginosa indicate that these proteins share conserved amino acid residues at positions known to be important for the binding of T4 AsiA to sigma70 region 4. We show that, like T4 AsiA, Rsd binds to sigma70 in a native protein gel and, as with T4 AsiA, a L18S substitution in Rsd disrupts this complex. Previous work has assigned sigma70 amino acid F563, within region 4.1, as a critical determinant for AsiA binding. This residue is also involved in the binding of sigma70 to the beta-flap of core, suggesting that AsiA inhibits transcription by disrupting the interaction between sigma70 region 4.1 and the beta-flap. We find that as with T4 AsiA, the interaction of KVP40 AsiA, Rsd, or AlgQ with sigma70 region 4 is diminished by the substitution F563Y. We also demonstrate that like T4 AsiA and Rsd, KVP40 AsiA inhibits transcription from sigma70-dependent promoters. We speculate that the phage AsiA orthologs, Rsd, and AlgQ are members of a related family in T4-type phage and bacteria, which interact similarly with primary sigma factors. In addition, we show that even though a clear MotA ortholog has not been identified in the KVP40 genome and the phage genome appears to lack typical middle promoter sequences, KVP40 AsiA activates transcription from T4 middle promoters in the presence of T4 MotA. We speculate that KVP40 encodes a protein that is dissimilar in sequence, but functionally equivalent, to T4 MotA.