The locations of the fertility inhibition genes finO and finP of the F-like conjugative multiple antibiotic-resistance plasmid R6-5 have been determined. As found previously for that of the fertility plasmid F, the finP gene of R6-5 is located close to the origin of DNA transfer, oriT, and to the promoter-proximal segment of the tra operon. Thus, finP is close to the site of action of the FinOP fertility inhibition system. In contrast, the finO gene is located on the other side of the tra operon, greater than 35 kilobases from the finP gene; finO is very close to the origin of vegetative replication, oriV, and to cistrons encoding functions involved in autonomous plasmid replication and plasmid incompatibility. A 4.5-kilobase fragment of R6-5 DNA containing the finO gene has been cloned on the high-copy amplifiable vector plasmid pBR322. This hybrid plasmid, designated pKTO31, causes severe repression of conjugal transfer of plasmid F, indicating the production of high cellular levels of finO protein. Two independent finO mutant derivatives were obtained after mutagenesis of the pKTO31 plasmid. Comparison of proteins synthesized by minicells carrying finO(-) mutant plasmids with those carrying various finO(+) plasmids enables the finO gene product to be tentatively identified as a 22,000-dalton protein.