Bacteriocins are ribosomally synthesized antibacterial peptides secreted by certain types of bacteria and are active against both closely related species,and members of other species. Currently bacteriocins are recommended for use as antimicrobial agents in the food industry and in medicine. Bacteriocin production significantly depends on several factors, such as culture conditions – pH, temperature and composition of the growth medium. Bacteriocins can both be released in the culture medium, and remain attached to the producer cell. The optimization of growth conditions for bacteriocin production and the increase of its activity are of great economic importance to reduce its production cost. The aim of this study was to determine the localization of a bacteriocin of Lactobacillus plantarum 42, active against Enterococcus faecalis, and to determine the optimal culture conditions in which its maximum output can be observed. The Lactobacillus plantarum 42 strain synthesizes bacteriocin, which is active against Enterococcus faecalis, which is released into a solid and a liquid nutrient medium, but in MRS Broth it is found only with tenfold concentration. Bacteriocin is detected at early stationary growth phase (18 hours) and remains active until 76 hours after initiation of fermentation. Max quantity of bacteriocin was detected after 48 hours of fermentation with the initial pH value of 6. There was no difference in the cultivation temperatures of 30oC and 37oC. This bacteriocin proved to be a secondary metabolite.