The primase-dependent priming signals, G sites, are directly recognized by the Escherichia coli primase (dnaG gene product) and conduct the synthesis of primer RNAs. In nucleotide sequence and secondary structure, there is no striking resemblance between the phage- and plasmid-derived G sites, except for the limited sequence homology near the start position of primer RNA synthesis. In this study, we analyzed the structure and function of a G site of plasmid R100, G site (R100), and discovered the necessity of the coexistence of two domains (domains I and III), which contains blocks A, B, and C, which are nucleotide sequences highly conserved among the plasmid- derived G sites. However, neither the internal region, domain II, between domains I and III nor the potential secondary structure proposed by Bahk et al. (J. D. Bahk, N. Kioka, H. Sakai, and T. Komano, Plasmid 20:266-270, 1988) is essential for single-stranded DNA initiation activity. Furthermore, chimeric G sites constructed between a G site of phage G4, G site (G4), and G site (R100) maintained significant single-stranded DNA initiation activities. These results strongly suggest that phage- and plasmid-derived G sites have functionally equivalent domains. The primase-dependent priming mechanisms of phage- and plasmid-derived G sites are discussed.