Several inositol sphingophospholipids (ISPL) were isolated from mycelia ofPhytophthora capsici, a phytopathogenic fungus of pepper (Capsicum annuum c.v. Yolo Wonder). The sphingophospholipids induce a protective response by cotyledons of young peppers against necrotic lesions caused by the pathogenP. capsici. The ISPL structures were determined by chemical methods, fast atom bombardment mass spectrometry and by fast atom bombardment collision-induced tandem mass spectrometry. All ISPL consisted of ceramides linked to inositol phosphate. The amidelinked fatty acids were found to have 16, 20 and 22 carbon chains. The long chain bases were identified as trimethylsilyl derivatives and shown to be C16-sphingosine and C16-dihydrosphingosine. The protective effect of the ISPL on cotyledons of young peppers against necrotic lesions caused by the pathogenP. capsici was shown not to be dependent on the unsaturation of the C16-sphingosine base and the nature of the acyl chain. We also investigated the effect of growth conditions on the ISPL produced in three different, chemically defined media that are commonly used for fungi cultures. We showed thatP. capsici synthesized one major class of compounds with molecular weights of Mr=833 and 835 containing the 16∶1 base and the 22∶1 or the 22∶0N-acyl group, independent of the growth medium used. The presence of phosphite or fosetyl-A1 did not affect the result.