Singlet O2, a metastable state of normal triplet O2, was identified as the cytotoxic agent that is probably responsible for in vitro inactivation of TA-3 mouse mammary carcinoma cells following incorporation of hematoporphyrin and exposure to red light. This photodynamic inactivation can be completely inhibited by intracellular 1,3-diphenylisobenzofuran. This very efficient singlet O2 trap is not toxic to the cells, nor does it absorb the light responsible for hematoporphyrin activation. The singlet O2-trapping product o-dibenzoylbenzene is formed nearly quantitatively intracellularly when the furan and hematoporphyrin are present during illumination but not when only the furan is present during illumination. The protective effect against photodynamic inactivation of TA-3 cells afforded by 1,3-diphenylisobenzofuran coupled with the nearly quantitative formation of the singlet O2-trapping product indicates that singlet O2 is the probable agent responsible for toxicity in this system.