Abstract: Saccharomyces cerevisiae produces the sphingolipid ceramide from de novo synthesis as well as hydrolysis of complex sphingolipids by the inositolphosphoceramide-phospolipase C, Isc1p, which is homologous to mammalian neutral sphingomyelinases. Though roles of sphingolipids in yeast stress responses are well characterized, whether Isc1p contributes to stress-induced sphingolipids has been unclear. The current study was undertaken in order to distinguish the relative roles of de novo sphingolipid biosynthesis versus Isc1p-mediated sphingolipid production in the heat stress response. Ceramide production was measured at normal and increased temperature in an ISC1 deletion and its parental strain. The results showed that Isc1p contributes specifically to the formation of the C 24 , C 24:1, and C 26 dihydroceramide species. The interaction between these two pathways of sphingolipid production was confirmed by the finding that ISC1 deletion is synthetically lethal with the lcb1-100 mutation. Interestingly, Isc1p did not contribute significantly to transient cell cycle arrest or growth at elevated temperature, responses known to be regulated by the de novo pathway. In order to define specific contributions of ISC1, microarray hybridizations were performed, and analyses showed misregulation of genes involved in carbon source utilization and sexual reproduction, which was corroborated by defining a sporulation defect of the isc1Delta strain. These results indicate that the two pathways of ceramide production in yeast interact, but differ in their regulation of ceramides of distinct molecular species and serve distinct cellular functions.