The IncN plasmid pKM101 (a derivative of R46) encodes the antirestriction protein ArdB (alleviation of restriction of DNA) in addition to another antirestriction protein, ArdA, described previously. The relevant gene, ardB, was located in the leading region of pKM101, about 7 kb from oriT. The nucleotide sequence of ardB was determined, and an appropriate polypeptide was identified in maxicells of Escherichia coli. Like ArdA, ArdB efficiently inhibits restriction by members of the three known families of type I systems of E. coli and only slightly affects the type II enzyme, EcoRI. However, in contrast to ArdA, ArdB is ineffective against the modification activity of the type I (EcoK) system. Comparison of deduced amino acid sequences of ArdA and ArdB revealed only one small region of similarity (nine residues), suggesting that this region may be somehow involved in the interaction with the type I restriction systems. We also found that the expression of both ardA and ardB genes is controlled jointly by two pKM101-encoded proteins, ArdK and ArdR, with molecular weights of about 15,000 and 20,000, respectively. The finding that the sequences immediately upstream of ardA and ardB share about 94% identity over 218 bp suggests that their expression may be controlled by ArdK and ArdR at the transcriptional level. Deletion studies and promoter probe analysis of these sequences revealed the regions responsible for the action of ArdK and ArdR as regulatory proteins. We propose that both types of antirestriction proteins may play a pivotal role in overcoming the host restriction barrier by self-transmissible broad-host-range plasmids. It seems likely that the ardKR-dependent regulatory system serves in this case as a genetic switch that controls the expression of plasmid-encoded antirestriction functions during mating.