Many Gram-positive and Gram-negative bacteria possess natural competence mechanisms for DNA capture and internalization. In Bacillus subtilis, natural competence is absolutely dependent upon the presence of a seven-gene operon known as the comG operon (comGA-G). In species of Streptococcus, this function has been described for a four-gene operon (comYA-D in Streptococcus gordonii and cglA-D in Streptococcus pneumoniae). In this study, a nine-orf operon (named comYA-I) required for natural competence in Streptococcus mutans was identified and characterized. Orf analysis of this operon indicates that the first four Orfs (ComYA-D) share strong homology with ComYA-D of S. gordonii and CglA-D of S. pneumoniae, the fifth to seventh Orfs (ComYE-G) match conserved hypothetical proteins from various species of Streptococcus with ComYF possessing a predicted ComGF domain, the eighth Orf (ComYH) shows a strong homology to numerous DNA methyltransferases from restriction/modification systems, and the ninth Orf (ComYI) is homologous to acetate kinase (AckA). RT-PCR analysis of the orf junctions confirmed that all nine orfs were present in a single transcript, while real-time RT-PCR analysis demonstrated that these orfs were expressed at a level very similar to that of the first orf in the operon. Mutations were constructed in all nine putative orfs. The first seven genes (comYA-G) were found to be essential for natural competence, while comYH and comYI had reduced and normal natural competence ability, respectively. Analyses of S. mutans comY-luciferase reporter fusions indicated that comY expression is growth-phase dependent, with maximal expression at an OD(600) of about 0.2, while mutations in ciaH, comC and luxS reduced the level of comY expression. In addition, comY operon expression appears to be correlated with natural competence ability.