In the presence of exciting light, iron and reductants, the singlet oxygen (¹O₂)-generating sensitizer protoporphyrin IX (PpIX) induces free radical lipid peroxidation in membranes, but gradually degrades in the process. We postulated that NO, acting as a chain-breaking antioxidant, would protect PpIX against degradation and consequently prolong its ability to produce ¹O₂. This idea was tested by irradiating PpIX-containing liposomes (LUVs) in the presence of iron and ascorbate, and monitoring the cholesterol hydroperoxides 5α-OOH and 7α/β-OOH as respective ¹O₂ and free radical reporters. 5α-OOH accumulation, initially linear with light fluence, slowed progressively after prolonged irradiation, whereas 7α/β-OOH accumulation only accelerated after an initial lag. The active, but not spent, NO donor spermine NONOate (0.4 mM) virtually abolished 7α/β-OOH buildup as well as 5α-OOH slowdown. Increasing membrane phospholipid unsaturation hastened the onset of rapid chain peroxidation and 5α-OOH slowdown. Accompanying the 5α-OOH effect was a steady decrease in ¹O₂ quantum yield and PpIX fluorescence at 632 nm, both of which were inhibited by NO. An NO-inhibitable decay of PpIX fluorescence was also observed during dark incubation of 5α-OOH–bearing LUVs with iron and ascorbate, confirming a link between chain peroxidation and PpIX loss. By protecting PpIX in irradiated membranes, NO might select for and prolong purely ¹O₂-mediated damage. Supporting this was our observation that ¹O₂-mediated photoinactivation of a nonmembrane target, lactate dehydrogenase, slowed concurrently with 5α-OOH accumulation and that spermine NONOate prevented this. Thus, NO not only protected membrane lipids against PpIX-sensitized free radical damage, but PpIX itself, thereby extending its ¹O₂-generating lifetime. Consistent findings were obtained using porphyrin-sensitized COH-BR1 cells. These previously unrecognized effects of NO could have important bearing on 5-aminolevulinate-based photodynamic therapy in which PpIX is metabolically deposited in tumor cells.