The sequence of an 897 base-pair fragment (42.1 to 43.0 kilobase co-ordinates on the F genetic map) containing the primary origin (ori-1) of mini-F replication has been determined. It contains one significant open reading frame, which probably codes for part of the C protein thought to be necessary for ori-1 replication activity. Tests of the ability of the sequenced ori-1 region to direct replication of DNA polymerase I-dependent replicons revealed that ori-1 replication requires adjacent mini-F sequences, 43.0 to 43.9 kilobase co-ordinates on the F genetic map in cis as well as a trans-acting gene product, probably the E protein, from the essential replication region of mini-F. In addition, a sequence required for control of pif gene expression has been mapped to a 160 base-pair region immediately upstream from the C (pifC) gene, and the crossover site of a specific recA-independent recombination mechanism has been mapped to a 220 base-pair region on the side of the pif control sequence distal to the C gene.