In the yeast Saccharomyces cerevisiae, we have demonstrated a necessary role for sphingolipids in the heat stress response through inhibition of nutrient import (Chung, N., Jenkins, G. M., Hannun, Y. A., Heitman, J., and Obeid, L. M. (2000) J. Biol. Chem. 275, 17229-17232). In this study, we used a combination of pharmacological and genetic approaches to determine which endogenous sphingolipid is the likely mediator of growth inhibition. When cells were treated with exogenous phytosphingosine (PHS, 20 microm) or structurally similar or metabolically related molecules, including 3-ketodihydrosphingosine, dihydrosphingosine, C(2)-phytoceramide (PHC), and stearylamine, only PHS inhibited growth. Also, PHS was shown to inhibit uptake of uracil, tryptophan, leucine, and histidine. Again this effect was specific to PHS. Because of the dynamic nature of sphingolipid metabolism, however, it was difficult to conclude that growth inhibition was caused by PHS itself. By using mutant yeast strains defective in various steps in sphingolipid metabolism, we further determined the specificity of PHS. The elo2Delta strain, which is defective in the conversion of PHS to PHC, was shown to have slower biosynthesis of ceramides and to be hypersensitive to PHS (5 microm), suggesting that PHS does not need to be converted to PHC. The lcb4Delta lcb5Delta strain is defective in the conversion of PHS to PHS 1-phosphate, and it was as sensitive to PHS as the wild-type strain. The syr2Delta mutant strain was defective in the conversion of DHS to PHS. Interestingly, this strain was resistant to high concentrations of DHS (40 microm) that inhibited the growth of an isogenic wild-type strain, demonstrating that DHS needs to be converted to PHS to inhibit growth. Together, these data demonstrate that the active sphingolipid species that inhibits yeast growth is PHS or a closely related and yet unidentified metabolite.