Cells ofStreptococcus lactis(354/07) synthesized and retained nisin when grown in a complex medium with 2.5% glucose at a constant pH of 6.7. Nisin was extracted from cells by a previously used method with hot 0.05 NHCl but milder methods of extraction from whole and broken cells using a variety of solvents were also tested. In the preferred method broken cells were extracted with 0.05 NHCl at 2 °C. The Cl−ions of the extract were exchanged for acetate on columns of the resin Amberlite CG 4B and the eluate was concentrated by acetone precipitation at −19 °C. The nisin was finally purified by pH gradient elution from CM cellulose columns. Three peaks with antibiotic activity were found, two of the peaks were minor and represented less than 5% of the nisin. The main peak gave a single band on electrophoresis. Electrophoresis of the material from the CM cellulose peaks revealed about 44 bands of basic proteins. Nisin made by the hot or cold HCl extraction behaved similarly in electrophoresis and CM cellulose chromatography but the antibiotic activity of the material isolated from the cold extract was nine times greater than that of the material isolated from the hot extract.