SUMMARY: Streptococcus lactis organisms grown in a glucose-containing medium pH maintained at pH 6.8 (neutral cocci) contained three- to seven-fold more nisin/unit dry weight than cocci grown in the same medium without pH control (acid cocci, terminal pH 4.2). After chemical fractionation of acid cocci 57% of the nisin was found in the fraction soluble in aqueous ethanol and 36% in a trypsin-insoluble residue. After fractionation of broken cocci (acid and neutral) by differential centrifugation up to 60% of the nisin was found in the 10,000 g sediment (walls). Nisin was also present in the 30,000 g sediment (membranes) and the 100,000 g sediment (ribosomes). The major difference between acid and neutral cocci was in the 100,000 g supernatant fluid (cell sap); cell sap from neutral cocci was 0.28% nisin whereas that from acid cocci was 0.04% nisin. Analysis of the cell wall indicated that it was composed of mucopeptide and polysaccharide. Teichoic acid could not be extracted. The polysaccharide was soluble in hot formamide and accounted for one-third of the dry weight of the wall; it contained rhamnose, galactose, glucose, glucosamine in the molar ratio of 5:1:1:1. Cell walls contributed 31% to the dry weight of acid cocci and 42% to the dry weight of neutral cocci.