Singlet oxygen (‍¹O₂) is a selective intermediate reactive oxygen species generated naturally in biological systems by light- and non-light mediated processes. Although ‍¹O₂ plays an important role in cell signaling and in maintaining homeostasis, it can be toxic due to its ability to diffuse across considerable distances. Several in vitro studies have investigated the pathways by which ‍¹O₂ mediates oxidation of biological molecules and potential pathogenesis. However, understanding how singlet oxygen exerts cell injury through the production of subsequent reactive oxygen species remains unexplored. To study this, we used a hydrophobic endoperoxide as a source of ‍¹O₂. Endoperoxides are reagents that quantitatively generate singlet oxygen in solution at 35°C by thermal decomposition. Our chemiluminescence and cell viability assay data revealed that ‍¹O₂ stimulated a secondary intracellular reactive oxygen species production in a very short time. To determine the source of these reactive oxygen species with endo­peroxide exposure, cells were treated with inhibitors targeting NADPH oxidases and platelet activating factor receptors. Our results showed that addition of the platelet activating factor receptor antagonist, Apafant (WEB2086), alleviated cell injury and hydrogen peroxide levels following endoperoxide stimulation. Furthermore, intracellular calcium assay data demonstrated a potential calcium sensitive production of intracellular reactive oxygen species.