Co-orientation of replication origins is the most common organization found in nature for multimeric plasmids. Streptococcus pyogenes broad-host-range plasmid pSM19035 and Escherichia coli pPI21 are among the exceptions. pPI21, which is a derivative of pSM19035 and pBR322, has two long inverted repeats, each one containing a potentially active ColE1 unidirectional origin. Analysis of pPI21 replication intermediates (RIs) by two-dimensional agarose gel electrophoresis and electron microscopy revealed the accumulation of a specific RI containing a single internal bubble. The data obtained demonstrated that initiation of DNA replication occurred at a single origin in pPI21. Progression of the replicating fork initiated at either of the two potential origins was transiently stalled at the other inversely oriented silent ColE1 origin of the plasmid. The accumulated RIs, containing an internal bubble, occurred as a series of stereoisomers with different numbers of knots in their replicated portion. These observations provide one of the first functional explanations for the disadvantage of head-to-head plasmid multimers with respect to head-to-tail ones.