The transfer systems of broad host range IncP plasmids are increasingly used in the genetic analysis and manipulation of many gram-negative bacteria. We have examined the structural and functional relatedness of the transfer origins of ten different broad host range plasmids which belong to the IncP incompatibility group. The data reported here, together with our results on relatedness of the replication segments of these plasmids, demonstrate that the genomes of all IncP plasmids share extensive sequence homology in the regions specifying the transfer origin and replication functions. The homology results reveal the existence of two subclasses among IncP plasmids, designated here as IncP alpha and IncP beta. Furthermore, a functional analysis of the transfer origins of IncP plasmids suggests strongly that the DNA-nicking relaxation complex (Guiney and Helinski 1979) is required for plasmid transfer during conjugation.