Ceramide is a key component of intracellular stress responses. Evidence is provided for a novel mechanism of ceramide formation that mediates solar ultraviolet (UV) A radiation-induced expression of the intercellular adhesion molecule (ICAM)-1. Similarly to UVA radiation, ceramide stimulation of human keratinocytes induced ICAM-1 mRNA expression and activated the ICAM-1 promoter through transcription factor AP-2. Ceramide-activated AP-2 and ceramide-induced ICAM-1 reporter gene activation were abrogated through deletion of the AP-2 binding site. UVA radiation increased the level of ceramide in keratinocytes and inhibition of sphingomyelin synthesis prevented UVA radiation-induced ICAM-1 expression. Hitherto, two pathways have been identified for ceramide accumulation: hydrolysis from sphingomyelin through neutral and acid sphingomyelinases, and de novo synthesis by ceramide synthase. UVA radiation did not activate any of these enzymes. Ceramide generation in UVA-irradiated cells, however, was inhibited by singlet oxygen quenchers and mimicked in unirradiated cells by a singlet oxygen-generating system. In addition, UVA radiation and singlet oxygen both generated ceramide in protein-free, sphingomyelin-containing liposomes. This study indicates that singlet oxygen triggers a third, non-enzymatic mechanism of ceramide formation.