The ether-lipid precursor sn-1-O-hexadecylglycerol (HG) can be used to compensate for early metabolic defects in ether-lipid biosynthesis. To investigate a possible metabolic link between ether-linked phospholipids and the rest of the cellular lipidome, we incubated HEp-2 cells with HG. Mass spectrometry analysis revealed major changes in the lipidome of HG-treated cells compared to that of untreated cells or cells treated with palmitin, a control substance for HG containing an acyl group instead of the ether group. We present quantitative data for a total of 154 species from 17 lipid classes. These species are those constituting more than 2% of their lipid class for most lipid classes, but more than 1% for the ether lipids and glycosphingolipids. In addition to the expected ability of HG to increase the levels of ether-linked glycerophospholipids with 16 carbon atoms in the sn-1 position, this precursor also decreased the amounts of glycosphingolipids and increased the amounts of ceramide, phosphatidylinositol and lysophosphatidylinositol. However, incubation with palmitin, the fatty acyl analogue of HG, also increased the amounts of ceramide and phosphatidylinositols. Thus, changes in these lipid classes were not ether lipid-dependent. No major effects were observed for the other lipid classes, and cellular functions such as growth and endocytosis were unaffected. The data presented clearly demonstrate the importance of performing detailed quantitative lipidomic studies to reveal how the metabolism of ether-linked glycerophospholipids is coupled to that of glycosphingolipids and ester-linked glycerophospholipids, especially phosphatidylinositols.