Photodynamic therapy, a novel cancer treatment using a photosensitive dye and visible light, produces an oxidative stress in cells, often leading to apoptotic cell death. Because ceramide is a second messenger that has been associated with stress-induced apoptosis, we investigated a possible link between photodynamic treatment (PDT), ceramide, and apoptosis in L5178Y-R (LY-R) cells. The cells undergo rapid apoptosis, initiating within 30 min of PDT. After a dose of PDT producing a 99.9% loss of clonogenicity, LY-R cells responded by an increased production of ceramide, which reached a maximum level in 60 min. For a constant light fluence and varying concentrations of the phthalocyanine photosensitizer Pc 4 [HOSiPcOSi(CH3)2(CH2)3N(CH3)2], the ED50 for ceramide generation (46 nM) was similar to the LD50 for clonogenic cell death (40 nM). We suggest that the PDT-stimulated increase in synthesis of ceramide in LY-R cells may be coupled to PDT-induced apoptosis. When the cells were exposed to exogenous N-acetyl-sphingosine (10 microM), apoptotic changes were observed only after 12-24 h. The delayed apoptotic response to the synthetic ceramide may be due to an induction of apoptosis by a different route than the one used by PDT.