A 7.5 kbp cryptic plasmid is found in almost all isolates of Chlamydia trachomatis. Real-time PCR assays, using TaqMan chemistry, were set up to quantify accurately both the chlamydial plasmid and the single copy, chromosomal omcB gene in the infectious, elementary bodies (EBs) of C. trachomatis L1 440. Plasmid copy number was also determined in the EBs of six other lymphogranuloma venereum (LGV) isolates (serovars L1-L3), ten trachoma isolates (serovars A-C) and nine urogenital isolates (serovars D-J). The results indicated an average plasmid copy number of 4.0+/-0.8 (mean+/-95 % confidence interval) plasmids per chromosome. During the chlamydial developmental cycle, up to 7.6 plasmids per chromosome were detected, indicating an increased plasmid copy number in the actively replicating reticulate bodies. Attempts to eliminate the plasmid from strain L1 440 using the plasmid-curing agents ethidium bromide, acridine orange or imipramine/novobiocin led to a paradoxical increase in plasmid copy number. It is speculated that the stress induced by chemical curing agents may stimulate the activity of plasmid-encoded replication (Rep) proteins. In contrast to C. trachomatis, only a single isolate of Chlamydophila pneumoniae bears a plasmid. C. pneumoniae strain N16 supports a 7.4 kbp plasmid in which ORF1, encoding one of the putative Rep proteins, is disrupted by a deletion and split into two smaller ORFs. Similar assay techniques revealed 1.3+/-0.2 plasmids per chromosome (mean+/-95 % confidence interval) in EBs of this strain. These findings are in agreement with the hypothesis that the ORF1-encoded protein is involved in, but not essential for, plasmid replication and control of copy number.