Conjugative plasmids have evolved entry exclusion mechanisms to inhibit redundant DNA transfer from donor cells into recipients harboring isogenic or closely related plasmids. This exclusion phenomenon has been documented in the incompatibility H group (IncH) plasmid R27. A cosmid library representing the majority of the large (180kb) R27 plasmid was transformed into recipient cells and a conjugation assay identified that an operon located in the conjugative transfer region 2 (Tra2) of R27, the Z operon, mediated entry exclusion in the IncH plasmid. Reverse-transcriptase analysis revealed that the Z operon is comprised of four genes, 015, eexB, 017, and eexA. Sub-cloning of the individual genes located within the Z operon and subsequent screening for the entry exclusion phenotype determined that two genes, eexA and eexB, independently inhibit the entry of IncH-related plasmids. Bacterial fractionation studies predominantly localized the EexA protein to the cytoplasmic membrane, and the EexB protein to the outer membrane. Recipient cells expressing EexA and EexB were unable to exclude the entry of R27 plasmids harboring mutations within the IncH entry exclusion genes eexA and eexB. The IncH entry exclusion proteins EexA and EexB likely prevent redundant plasmid transfer by interaction with one another.