Activation of transcription initiation by the cI protein of phage lambda is thought to be mediated by a direct interaction between cI and RNA polymerase at the PRM promoter.Two negatively charged amino acid residues in the DNA binding domain of cI play a key role in activation, suggesting that these residues contact RNA polymerase. The subunit of RNA polymerase involved was identified by selecting polymerase mutants that restored the activation function of a mutant form of cI protein. Although previous studies suggest that several activators interact with the alpha subunit of RNA polymerase, the results here suggest that cI interacts with the sigma subunit. An arginine to histidine change near the carboxyl terminus of sigma specifically suppresses an aspartic acid to asparagine change in the activation region of cI. This finding supports the direct-contact model and suggests that a cluster of positively charged residues near the carboxyl terminus of sigma is the target of the negatively charged activation region of cI.