Streptococcus pyogenes is capable of causing a variety of human diseases ranging from superficial or deep tissue infections to non-infectious post-streptococcal infection sequelae. In this paper, we report the use of a Tn917-lacZ transposon to isolate random lacZ transcription fusions in the S. pyogenes chromosome. Libraries of random Tn917-lacZ mutants were generated in a representative opacity factor positive strain CS101 (M49) and an opacity factor negative strain 1881 (M1). Several different mutant phenotypes were isolated. These included: temperature-regulated promoters, growth phase/cell density-regulated promoters and a human plasma-inducible promoter. Expression of the temperature-regulated fusions was 5-10-fold higher when grown at 30 degrees C compared to growth at 37 degrees C. The growth phase-regulated fusions were induced 30-fold at late exponential phase and were repressed by a diffusible S. pyogenes factor(s). Expression of the human plasma-inducible fusion was induced 10-15-fold by human plasma or sera, 4-fold by rabbit sera and was repressed by horse and mouse sera. In addition, hemolysin negative and capsule over expression mutants were isolated. These results demonstrate the utility of Tn917-lacZ mutagenesis for the identification of S. pyogenes promoters.