Pseudomonas aeruginosa is equipped with the Sec and Tat protein secretion systems, which translocate the xenobiotic transporter MexAB-OprM and the pathogenic factor phospholipase C (PlcH), respectively. When the signal sequence of MexA was replaced with that of PlcH, the hybrid protein was successfully expressed and recovered from the periplasmic fraction, suggesting that the hybrid protein had been translocated across the inner membrane. MexA-deficient cells harboring the plasmid carrying the plcH-mexA fusion gene showed antibiotic resistance comparable to that of the wild-type cells. This result suggested that MexA secreted via the Tat machinery was properly assembled and functioned as a subunit of the MexAB-OprM efflux pump. A mutation was introduced into the chromosomal tatC gene encoding an inner membrane component of the Tat protein secretion machinery in mexA-deficient cells, and they were transformed with the plasmid carrying the plcH-mexA fusion gene. The transformants showed antibiotic susceptibility comparable to that of mexA-deficient cells, indicating that the hybrid protein was not transported to the periplasm. Whole-cell lysate of the mexA-tatC double mutant harboring the plcH-mexA plasmid produced mainly unprocessed PlcH-MexA. The periplasmic fraction showed no detectable anti-MexA antibody-reactive material. On the basis of these results, we concluded that MexA could be translocated across the inner membrane through the Tat pathway and assembled with its cognate partners, MexB and OprM, and that this complex machinery was fully functional. This hybrid protein translocation system has the potential to be a powerful screening tool for antimicrobial agents targeting the Tat system, which is not present in mammalian cells.