The mechanism of Staphylococcus aureus inactivation by deuteroporphyrin (DT) and light was studied with singlet oxygen quenchers or hydroxyl radical scavengers. The light-activated DT (10 μ/ml) reduced the viability of the culture to less than 1%, whereas methionine, tryptophan, and 1,4-diazabicyclo-2,2,2-octane (DBCO) used as singlet oxygen quenchers provided almost 60% protection. Propylgallate, which is a hydroxyl free radical scavenger, also provided 60% protection. The presence of a singlet oxygen quencher and propylgallate provided almost complete protection from inactivation (96%). Photoinactivation in the absence of culture media (in saline) increased the killing rate and decreased the ability of the singlet oxygen quenchers to protect. In the same conditions damage from hydroxl free radicals was well protected by propyl gallate. The present results indicate that S. aureus photoinactivation by DT and light is mediated by both singlet oxygen and hydroxyl free radicals.