In this study we report the phototoxicity toward HaCaT keratinocytes that results from the photogeneration of superoxide and singlet oxygen (1O2) by four different “water-soluble” fullerene (C60) preparations—monomeric (γ-CyD)2/C60 (γ-cyclodextrin bicapped C60) and three aggregated forms—THF/nC60 (prepared by solvent exchange from THF solution); Son/nC60 (prepared by sonication of a toluene/water mixture); and γ-CyD/nC60 (prepared by heating the [γ-CyD]2/C60 aqueous solution). Our results demonstrate that all four C60 preparations photogenerate 1O2 efficiently. However, the properties of C60-generated 1O2, including its availability for reactions in solution, are markedly different for the monomeric and aggregated forms. 1O2 produced by monomeric (γ-CyD)2/C60 is quenchable by NaN3 and its quantum yield in D2O, which is only weakly dependent on oxygen concentration, is as high as C60 in toluene. In contrast, 1O2 generated from aggregated C60 is not quenchable by NaN3, exhibits a solvent-independent short-lived lifetime (ca 2.9 μs), is highly sensitive to oxygen concentration while its phosphorescence is redshifted. All these features indicate that 1O2 is sequestered inside the C60 aggregates, which may explain why these preparations were not phototoxic toward HaCaT cells. Electron paramagnetic resonance studies demonstrated the generation of the C60 anion radical (C60•−) when (γ-CyD)2/C60 was irradiated (λ > 300 nm) in the presence of a reducing agent (NADH); spin trapping experiments (λ > 400 nm) with 5,5-dimethyl-1-pyrroline N-oxide clearly showed the generation of superoxide resulting from the reaction of C60•− with oxygen. In vitro tests with HaCaT keratinocytes provided evidence that (γ-CyD)2/C60 phototoxicity is mainly mediated by 1O2 (Type II mechanism) with only a minor contribution from free radicals (Type I mechanism).