Abstract: Sphingoid long-chain base 1-phosphates (LCBPs) are widely conserved, bioactive lipid molecules. In yeast, LCBPs are known to be involved in several cellular responses such as heat shock resistance and Ca(2+) mobilization, although their target molecules and signaling pathways remain unclear. To identify genes involved in LCBP signaling and in regulation of LCBP synthesis, we performed transposon-mutagenesis in cells lacking the LCBP lyase DPL1 and LCBP phosphatase LCB3 genes, and screened for phytosphingosine (PHS)-resistant clones. Further isolation and identification revealed eight genes (PBP1, HEM14, UFD4, HMG1, TPS1, KES1, WHI2, and ERG5), in addition to the previously characterized LCB4 and PDR5 genes, that are involved in PHS resistance. Of these eight, four were ergosterol-related genes (HEM14, HMG1, KES1, and ERG5). We further demonstrated that protein expression of the long-chain base kinase Lcb4p was reduced in deltahem14 and deltahmg1 cells, likely a consequence of decreased activity of the heme-dependent transcription factor Hap1p. In addition, phosphorylation of Lcb4p was decreased in all the ergosterol-related mutants isolated and other ergosterol mutants constructed (deltaerg2, deltaerg3, and deltaerg6). Finally, plasma membrane localization of Lcb4p was found to be reduced in the deltaerg6 cells. These results suggest that changes in sterol composition affect the phosphorylation of Lcb4p due to the altered localization. The other genes isolated, PBP1, UFD4, TPS1, and WHI2, may be involved in the LCBP signaling.