SecY is an integral membrane protein, spanning the cytoplasmic membrane of E. coli probably 10 times and required for efficient translocation of other proteins across the membrane. We report here that this protein can be specifically cleaved at the central region of the polypeptide after cell disruption, and cytoplasmic membrane preparations often contain a degradation product of SecY. This cleavage was ascribed to the action of the outer membrane-associated protease specified by the ompT gene, since the cleavage was not observed in ompT-defective mutants. Thus, we propose that an ompT mutant should be used for in vitro analysis of protein translocation and the SecY protein. We mutated the ompT gene by insertion of a kanamycin resistance determinant to facilitate strain construction by P1 transduction.