Thomicide is a complex preparation including a bacteriocin-like substance. To localize the determinant responsible for synthesis of the bacteriocin-like substance, DNA of the streptococcus producing thomicide was isolated and studied. Equilibrium centrifugation of the total DNA preparation in the gradient of cesium chloride-ethidium bromide yielded DNA of one density. The total DNA preparation was obtained with alkaline and neutral lysis. Restriction analysis of the streptococcal DNA followed by electrophoretic separation in agarose gel in comparison to the DNA standards of lambda phage and plasmid pBR 322 confirmed the absence of the plasmid or any other extrachromosomal DNA. Chromosomal localization of the determinant encoding biosynthesis of the thomicide bacteriocin-like substance was shown.