The mechanisms by which gene products inhibit the conjugal transfer of IncP plasmids (e.g., RP1) have been little studied. We have isolated and characterized one such gene, fipA (624 nucleotides), from the SmaI (14.8 kb)-AatII (15.6 kb) region of pKM101(IncN). This gene, which is also conserved in other IncN plasmids, is transcribed in an anticlockwise direction, probably as part of a transfer operon that includes traHI. The FipA protein (24 kDa) appears to be cytoplasmic and, when expressed from a multicopy plasmid, retards the growth of Escherichia coli WP2. The mode of action of fipA was compared with that of the apparently unrelated pifC gene from F(IncFI). Both genes inhibit the transfer of IncPalpha and IncPbeta plasmids but to different degrees. They also inhibit the mobilization of RSF1010 (which requires the RP1 pilus genes and traG) but not of CloDF13 (which encodes a traG homolog). Evidence that traG was the specific target of inhibition was obtained in an artificial system in which cloned traG was used to enhance RSF1010 mobilization via the N pilus system. Such enhancement did not occur in the presence of fipA or pifC. The availability of an in vivo assay of PifC enabled us to show that F pif operon expression increased in cells carrying F'lac and traG, but only if the traG coding sequence was intact. This finding suggested that conjugal inhibition of RP1 was most likely due to a PifC-TraG protein interaction. On phenotypic grounds inhibition of traG by fipA is also likely to occur posttranscriptionally. Whether or not the selection of traG as the inhibition target is an evolutionary tactic to limit the spread of P plasmids, we anticipate that fipA and pifC will prove useful in further investigation of the conjugal roles of traG and its homologs.