For Serratia marcescens, secreted hemolysin/cytotoxin is not only secreted but also activated by an outer membrane protein. Excluding posttranslational processing by mass spectrometry, the conformation of active and inactive ShlA derivatives strongly differed in electrophoretic mobilities, gel permeation chromatography, sensitivity to trypsin, circular dichroism, and intrinsic fluorescence. We concluded that ShlB interacts with ShlA during secretion and imposes a conformational change in ShlA to form the active hemolysin.