Highest antimicrobial activity of peptide ST4SA (51,200AU/mL) was recorded after 14h of growth in MRS broth with optimal production at pH 6.0 or 6.5. Growth of strain ST4SA in the presence of tryptone, yeast extract, or a combination of the two, yielded 102,400AU/mL. An increase in production of peptide ST4SA to 102,400AU/mL was recorded in the presence of 20.0g/L fructose, but decreased to 25,600AU/mL in the presence of lactose (20.0g/L) or mannose (20.0g/L) as sole carbon source. Lower activity (25,600AU/mL) was recorded when 2.0g/L K 2 HPO 4 was replaced by 2.0g/L KH 2 PO 4 in MRS broth. An increase of K 2 HPO 4 to 10.0g/L and 20.0g/L resulted in higher activity (102,400AU/mL). Addition of glycerol to MRS broth had a negative effect on peptide ST4SA production. Production of peptide ST4SA required the presence of magnesium sulphate, manganese sulphate and 5.0g/L sodium acetate. Exclusion of tri-ammonium citrate from the medium resulted in reduction of activity to 3,200AU/mL. Maximum activity (102,400AU/mL) was recorded in MRS supplemented with 1.0ppm Vit. C, DL-6,8-thioctic acid or thiamine, respectively. Growth of Listeria ivanovii susbp. ivanovii ATCC 19119 in the presence of peptide ST4SA (12,800AU/mL) resulted in 99% cell lysis after 18h. Improved production of peptide ST4SA was recorded in MRS broth (Biolab) pre-treated with Amberlite XAD-1180. Precipitation with ammonium sulphate, followed by gel filtration chromatography, yielded the highest level of peptide ST4SA. This paper describes the partially deproteination of growth medium to facilitate peptide ST4SA purification.