ABSTRACT Type II (proteic) toxin-antitoxin systems (TAS) are ubiquitous among bacteria. In the chromosome of the pathogenic bacterium Streptococcus pneumoniae , there are at least eight putative TAS, one of them being the yefM-yoeB Spn operon studied here. Through footprinting analyses, we showed that purified YefM Spn antitoxin and the YefM-YoeB Spn TA protein complex bind to a palindrome sequence encompassing the −35 region of the main promoter (P yefM2 ) of the operon. Thus, the locus appeared to be negatively autoregulated with respect to P yefM2 , since YefM Spn behaved as a weak repressor with YoeB Spn as a corepressor. Interestingly, a BOX element, composed of a single copy (each) of the boxA and boxC subelements, was found upstream of promoter P yefM2 . BOX sequences are pneumococcal, perhaps mobile, genetic elements that have been associated with bacterial processes such as phase variation, virulence regulation, and genetic competence. In the yefM-yoeB Spn locus, the boxAC element provided an additional weak promoter, P yefM1 , upstream of P yefM2 which was not regulated by the TA proteins. In addition, transcriptional fusions with a lacZ reporter gene showed that P yefM1 was constitutive albeit weaker than P yefM2 . Intriguingly, the coupling of the boxAC element to P yefM1 and yefM Spn in cis (but not in trans ) led to transcriptional activation, indicating that the regulation of the yefM-yoeB Spn locus differs somewhat from that of other TA loci and may involve as yet unidentified elements. Conservation of the boxAC sequences in all available sequenced genomes of S. pneumoniae which contained the yefM-yoeB Spn locus suggested that its presence may provide a selective advantage to the bacterium.