Whether slo, the gene encoding streptolysin O (SLO), a streptococcal cytolysin, has its own promoter or not is unsettled as yet. Present analyses demonstrate that slo is a member of an operon covering the upper-stream nusG and nga (NADase) genes, from which transcription of slo proceeds polycistronically, and major transcript is produced by readthrough from nga promoter. Mutational conversion of the sixth nucleotide T at the putative -10 region of chromosomal nga gene into C caused a drastic decrease in both NADase and SLO activities and the disappearance of the two corresponding mRNA bands from the Northern blot profile. The initiation site of the transcription was determined at 56 bp upstream (NusG gene) and 25 bp upstream (NADase gene) of each initiation codon. Although the promoter region of slo gene is highly conserved between group A and C streptococci, the proper slo promoter is nonfunctional in group C strain H46A. Moreover, commonly conserved arrangement was limited to the nusG-nga-orf1-slo region. These results indicate an intimate relationship between NADase and SLO in the regulation of their biosynthesis. Additional results suggest that NADase, synthesized as precursor with feeble activity, is activated by removing the carboxyl terminal region during or after secretion into culture medium.