A DNA sequence consisting of 617 base pairs (bp) from the region of the origin of replication of the broad-host range plasmid RK2 has been determined. Included within this sequence is a 393 bp HpaII restriction fragment that provides a functional origin or replication when other essential RK2 specified functions are provided in trans. Also contained in this sequence is a region, distinguished functionally from the replication origin, which is involved in the expression of inc2 incompatibility, i.e., the ability of derivatives of RK2 to eliminate a resident RK2 plasmid. The 617 bp sequence includes eight 17 base pair direct repeats with 5 located within the region required for a functional replication origin and 3 within the region involved in inc2 incompatibility. In addition, a 40 bp region rich in A-T followed by a 60 bp stretch having a high G + C content is present. Deletion evidence indicates that the A-T rich and possibly the G + C regions are required for a functional replication origin. Based on the evidence contained in this and the preceding paper (Thomas et al. 1980 b) a model will be presented for the involvement of these specific sequences in the initiation of RK2 DNA replication, plasmid maintenance and plasmid incompatibility.