Plasmid pT181 DNA secondary structures have been analyzed in vitro by nuclease S1 digestion and in vivo by bromoacetaldehyde treatment. A cruciform structure occurring at the pT181 replication origin in vitro is greatly enhanced by the binding of the plasmid-encoded initiator protein RepC. In vivo a DNA secondary structure also existed in the replication origin. Its frequency of formation was correlated with efficiency of RepC utilization. These data suggest that cruciform extrusion at the origin is involved in initiation of pT181 rolling-circle replication. A neighboring DNA structure influences the conformation of the origin in vivo.