In this study we report the phototoxicity toward HaCaT keratinocytes that results from the photogeneration of superoxide and singlet oxygen ((1)O(2)) by four different "water-soluble" fullerene (C(60)) preparations-monomeric (gamma-CyD)(2)/C(60) (gamma-cyclodextrin bicapped C(60)) and three aggregated forms-THF/nC(60) (prepared by solvent exchange from THF solution); Son/nC(60) (prepared by sonication of a toluene/water mixture); and gamma-CyD/nC(60) (prepared by heating the [gamma-CyD](2)/C(60) aqueous solution). Our results demonstrate that all four C(60) preparations photogenerate (1)O(2) efficiently. However, the properties of C(60)-generated (1)O(2), including its availability for reactions in solution, are markedly different for the monomeric and aggregated forms. (1)O(2) produced by monomeric (gamma-CyD)(2)/C(60) is quenchable by NaN(3) and its quantum yield in D(2)O, which is only weakly dependent on oxygen concentration, is as high as C(60) in toluene. In contrast, (1)O(2) generated from aggregated C(60) is not quenchable by NaN(3), exhibits a solvent-independent short-lived lifetime (ca 2.9 micros), is highly sensitive to oxygen concentration while its phosphorescence is redshifted. All these features indicate that (1)O(2) is sequestered inside the C(60) aggregates, which may explain why these preparations were not phototoxic toward HaCaT cells. Electron paramagnetic resonance studies demonstrated the generation of the C(60) anion radical (C(60)) when (gamma-CyD)(2)/C(60) was irradiated (lambda > 300 nm) in the presence of a reducing agent (NADH); spin trapping experiments (lambda > 400 nm) with 5,5-dimethyl-1-pyrroline N-oxide clearly showed the generation of superoxide resulting from the reaction of C(60) with oxygen. In vitro tests with HaCaT keratinocytes provided evidence that (gamma-CyD)(2)/C(60) phototoxicity is mainly mediated by (1)O(2) (Type II mechanism) with only a minor contribution from free radicals (Type I mechanism).