The bactericidal effect of several major types of antibiotics has recently been demonstrated to be dependent on the formation of toxic amounts of hydroxyl radicals (OH) resulting from oxidative stress in metabolically active cells. Since killing by the antimicrobial peptide colistin does not require bacterial metabolic activity, we tested whether the bactericidal effect of colistin depends on the formation of OH. In Pseudomonas aeruginosa cultures, OH-mediated killing by ciprofloxacin was demonstrated by decreased bacterial survival and induction of 3'-(p-hydroxyphenyl) fluorescein (HPF) fluorescence. OH-mediated killing by ciprofloxacin was further confirmed by rescue of cells and reduction of HPF fluorescence due to prevention of OH accumulation by scavenging with thiourea, by chelating with dipyridyl, by decreasing metabolism as well as by anoxic growth. In contrast, no formation of OH was seen in P. aeruginosa during killing by colistin, and prevention of OH accumulation could not rescue P. aeruginosa from killing by colistin. These results therefore demonstrate that the bactericidal activity of colistin on P. aeruginosa is not dependent on oxidative stress. In conclusion, antimicrobial peptides that do not rely on OH formation should be considered for treatment of Gram-negative bacteria growing at low oxygen tension such as in endobronchial mucus and paranasal sinuses in cystic fibrosis patients, in abscesses and in infectious biofilm.