pIP501 is a broad-host-range plasmid originating from Streptococcus agalactiae. In this report we show that (i) it replicates by a theta mechanism initiating at the 3' end of the gene encoding the replication protein RepR and progressing in the direction of transcription of this gene; (ii) its replication origin lies within or a few nucleotides downstream from ORF R and not upstream from it as suggested in the literature (Brantl et al. (1990) Nucleic Acids Res. 18, 4783-4789); (iii) the RepR protein positively regulates pIP501 copy number; and (vi) the main function ensured by the sequences located upstream from ORF R is to express this ORF. Since the replication properties of pIP501 are indistinguishable from those of the highly related Enterococcus faecalis plasmid pAM beta 1, we conclude that these elements form the first family of theta replicating plasmids in gram-positive bacteria. Based on sequence similarities, we extend this family to the S. pyogenes plasmid pSM19035 and to 12 other plasmids isolated from streptococci or enterococci.