A recent model for cytolysin-mediated translocation in Streptococcus pyogenes proposes that NAD-glycohydrolase is translocated through streptolysin O-generated pores into a host cell (J. Madden, N. Ruiz, and M. Caparon, Cell 104:143-152, 2001). This model also assumes that the NAD-glycohydrolase (nga) and streptolysin O (slo) genes that code for these products are organized in an operon-like structure expressed from a single promoter only (nga). We expand this model by showing that slo possesses its own autonomous promoter, which is located 155 bp upstream of the slo gene. Under experimental conditions in which S. pyogenes is grown in THY medium, the strength of the slo promoter, as measured by the activity of a lacZ reporter gene, resulted in low but highly reproducible values. Finally, we demonstrated that sloR, a S. pyogenes gene that closely resembles the Clostridium perfringens pfoR gene, exerts a negative effect on the expression of the slo gene.