Plasmids of the incompatibility groups HI and HII (IncH plasmids) generally confer multiple antibiotic resistances upon their host pathogenic strain. IncHI group plasmids are distinguished by their property of optimal transfer by conjugation at temperatures below 30 degreesC. allowing for the spread of multiple antibiotic resistance outside their host natural environment, the gut. Plasmids of the IncHI1 subgroup encode multiple replicons. In the present study it is shown that the prototype lncHI2 subgroup plasmid, R478, contains at least two iteron-controlled autoreplicative regions, RepHI2A and RepHI1A((R478)). The DNA sequence and the molecular characteristics of each replicon region are described. RepH12A is unique to plasmids of the IncHI2 subgroup and contains an unusually large number of iteron sequences downstream of the replication initiator gene. The nucleotide sequence of the replication initiator gene and of the iterons within RepHI1A((R478)) show very close similarity with those of the previously reported RepHI1A replicon of the IncHI1 subgroup plasmid R27, The presence of RepHI1A((R478)) on R478 most likely accounts for the observed incompatibility between R478 and plasmids of the IncHI1 subgroup. These are the first autoreplicative regions from an IncHI2 subgroup plasmid to be described.