Hemolytic Escherichia coli cells were found to synthesize and secrete significant amounts of hemolysin into a mineral salt-glucose medium containing hemoglobin. The release of de novo-synthesized hemolysin was stopped in the presence of energy metabolism inhibitors such as 2,4-dinitrophenol, sodium azide, or potassium cyanide, resulting in an accumulation of intracellular hemolysin. A similar effect was observed in the presence of procaine, a neuroactive drug which inhibits the processing of exoproteins. Small amounts of hemolysin were secreted into the medium within approximately 10 min of inhibition of protein synthesis by chloramphenicol. This represented the final release of preformed periplasmic hemolysin en route to secretion through the outer membrane and was not caused by adsorption of external hemolysin to the cell surface. This secretion was not energy dependent but was inhibited above pH 8 and at low temperatures (10 to 20 degrees C). We concluded that two transport processes are involved in hemolysin secretion. De novo-synthesized hemolysin is extruded by an energy-dependent process through the cytoplasmic membrane and probably requires processing. In the periplasmic space a small internal pool of preformed hemolysin is accumulated temporarily before being transported through the outer membrane. Release of hemolysin through the outer membrane does not require energy or de novo protein synthesis.