Abstract: Dental caries is a bacterial infection of the dentition. The acidic byproduct of bacterial carbohydrate fermentation demineralizes tooth structures, resulting in cavitations. One of the pathogens implicated in dental caries is Streptococcus mutans. Its virulence is enhanced by its production of bacteriocins, mutacins, which inhibit the growth of other endogenous bacteria. It is suspected that mutacin production helps S. mutans to predominate in the oral biofilm. Studies on mutacin regulation in S. mutans can provide insight into the process of S. mutans colonization and caries progression, thus aiding in the development of preventive, diagnostic and therapeutic modalities for dental caries. The goal of this study is to use a random insertional mutagenesis approach to reveal genes which are involved in mutacin I production in the virulent strain UA140. A random insertional mutagenesis library was constructed and screened for mutacin I defective phenotypes. Mutacin I defective clones were isolated and their insertion sites were determined by PCR amplification or plasmid rescue followed by sequencing. Twenty five genes were identified and they can be categorized into the following functional classes: two-component sensory systems, stress responses, energy metabolism and central cellular processes. Several conserved hypothetical proteins were also identified. Amongst these potential mutacin regulators, six genes were further analyzed based on their putative functions. Real-Time RT-PCR analyses of these candidate genes revealed transcriptional control on mutR, the transcriptional regulator of mutacin I and mutA, the structural gene for mutacin I. The function of covRSX operon, was further analyzed because of its involvement in multiple cellular functions. Defined genetic and microarray analyses of the covRS TCS provided new insights into the relationship between CovR, the response regulator, CovS, the histidine kinase sensor and CovX, a conserved hypothetic protein in the operon. In addition, some of these candidate genes seemed to exert a transcriptional effect on a luxS related transcription regulator, IrvA. To uncover the relationship of these genes, epistatic studies were performed. The results demonstrate that most of these genes are involved in parallel pathways leading to mutacin expression, which suggests that mutacin I production is affected stringently by diverse and complex regulatory pathways.