Viable Neisseria gonorrhoeae exposed to streptonigrin generate intracellular hydroxyl radical detected by spin-trapping with 5,5-dimethyl-l-pyrroline-N-oxide; gonococci exposed to paraquat generate primarily superoxide (J. Biol. Chem., 262: 13404-143048, 1987). The use of streptonigrin and paraquat provide a model with which to examine the action and site(s) of hydroxyl radical-mediated damage. N. gonorrhoeae exposed to streptonigrin, but not paraquat, developed extensive chromosomal, plasmid, and RNA damage. Addition of excess Fe+3 to the reaction mixture enhanced intracellular hydroxyl radical formation by paraquat, detectable as DNA damage. Desferal and dimethyl sulfoxide allowed approximately 25% of protection of plasmid DNA damage as judged by linear scanning densitometry. These results demonstrate DNA and RNA damage in viable organisms exposed to intracellular redox stress and confirm the critical role of hydroxyl radical in this process.