The amount of lactocin S activity in a growing culture depends on the growth stage of the bacteria, the pH of the medium, the presence of ethanol, and the aeration of the culture. We observed the highest levels of bacteriocin activity in the early stationary growth phase of cultures at 30 degrees C. When Lactobacillus sake L45 was grown in a fermenter at pH 5, it produced 2,000 to 3,000 bacteriocin units per mi, which represented an 8- to 10-fold increase in bacteriocin production compared with production during batch culture fermentation. Less than 10% of this level of bacteriocin activity,vas observed during fermentation at pH 6.0. Where 1% ethanol,vas included in the growth medium, a two- to fourfold increase in the bacteriocin yield was observed. Aeratiug the culture during growth almost completely eliminated the production of active bacteriocin. Our results also showed that lactocin S-mediated killing of target cells depended on the pH of the culture. The pH had to be less than 6 in order to obtain a bactericidal effect with lactocin S-sensitive cells. At pH values greater than 6, lactocin S had no apparent effect on sensitive cells.