During industrial processes, the dairy organism Streptococcus thermophilus is exposed to stress conditions. Its ability to survive and grow in an aerobic environment indicates that it must possess defensive mechanisms against reactive oxygen species. To identify the genes involved in oxidative stress defence, a collection of mutants was generated by random insertional mutagenesis and screened for menadione sensitivity and resistance. Results obtained for resistant clones allowed the identification of eight loci. The insertions affected genes whose homologues in other bacteria were previously identified as being involved in stress response (deoB, gst) or transcription regulation (rggC) and five ORFs of unknown function. The tolerance of the eight mutants to air-exposure, methyl viologen and H 2 O 2 was studied. Real-time quantitative PCR was used to analyse the transcript level of mutated genes and revealed that most were down-regulated during oxidative stress.