It was demonstrated recently that the SeqA protein, a main negative regulator of Escherichia coli chromosome replication initiation, is also a specific transcription factor. SeqA specifically activates the bacteriophage lambda pR promoter while revealing no significant effect on the activity of another lambda promoter, pL. Here, we demonstrate that lysogenization by bacteriophage lambda is impaired in E. coli seqA mutants. Genetic analysis demonstrated that CII-mediated activation of the phage pI and paQ promoters, which are required for efficient lysogenization, is less efficient in the absence of seqA function. This was confirmed in in vitro transcription assays. Interestingly, SeqA stimulated CII-dependent transcription from pI and paQ when it was added to the reaction mixture before CII, although having little effect if added after a preincubation of CII with the DNA template. This SeqA-mediated stimulation was absolutely dependent on DNA methylation, as no effects of this protein were observed when using unmethylated DNA templates. Also, no effects of SeqA on transcription from pI and paQ were observed in the absence of CII. Binding of SeqA to templates containing the tested promoters occurs at GATC sequences located downstream of promoters, as revealed by electron microscopic studies. In contrast to pI and paQ, the activity of the third CII-dependent promoter, pE, devoid of neighbouring downstream GATC sequences, was not affected by SeqA both in vivo and in vitro. We conclude that SeqA stimulates transcription from pI and paQ promoters in co-operation with CII by facilitating functions of this transcription activator, most probably by allowing more efficient binding of CII to the promoter region.