Genetic characterization of a Tn916 transposon mutant, Streptococcus mutans TS-1, defective in mutacin II production, revealed that the transposon was inserted into the 3' region of a diacylglycerol kinase (dgk) gene. The insertion occurred in the same region as described for another S. mutans mutant, GS5Tn1, which was altered in its ability to respond to environmental stress (Y. Yamashita, T. Takehara, and H. K. Kuramitsu, J. Bacteriol. 175:6220-6228, 1993). Quantitative primer extension from the mutacin structural gene mutA showed a decreased level (about eightfold) of mut A transcription for mutant T8-1. Mutacin production was restored by transforming mutant T8-1 with integration vector pVA891 containing an intact dgk gene. These data indicated that the full-length dgk gene product along with the mutacin biosynthetic operon are required for the production of the mutacin II lantibiotic.