We have surveyed the frequency of each of 64 trinucleotide permutations at every nucleotide frame located from 1 to 15 nucleotides upstream of primer RNA-DNA transition sites mapped within a 1.5 kb region of the bacteriophage lambda genome and a 1.4 kb region of the Escherichia coli genome. We have demonstrated that in both systems initiation of DNA synthesis strongly correlates with a CAG sequence located 11 nucleotides upstream of the DNA start sites. Based on the examination of various reports of the priming reaction catalyzed by E. coli primase in vivo and in vitro, we propose that (i) E. coli primase itself recognizes a 3'GTC 5' sequence on the template strand, (ii) DnaB helicase releases the specificity of E. coli primase and, (iii) the consensus recognition sequence for E. coli primase associated with DnaB helicase is 3'PuPyPy 5'.