Lipid species changes for SV40-transformed fibroblasts from wild-type or from diacylglycerol kinase-ε (DGKε) or diacylglycerol kinase-α (DGKα) knockout mice were determined for glycerophospholipids, polyphosphatidylinositides (GPInsP_n) and diacylglycerol (DAG) using direct infusion mass spectrometry. Dramatic differences in arachidonate (20:4 fatty acid)-containing lipids were observed for multiple classes of glycerophospholipids and polyphosphatidylinositides between wild-type and DGKε knockout cells. However, no difference was observed in either the amount or the acyl chain composition of DAG between DGKε knockout and wild-type cells, suggesting that DGKε catalyzed the phosphorylation of a minor fraction of the DAG in these cells. The differences in arachidonate content between the two cell lines were greatest for the GPInsP_n lipids and lowest for DAG. These findings indicate that DGKε plays a significant role in determining the enrichment of GPInsP_n with 20:4 and that there is a pathway for the selective translocation of arachidonoyl phosphatidic acid from the plasma membrane to the endoplasmic reticulum. In contrast, no substantial difference was observed in the acyl chain composition of any class of glycerophospholipid or diacylglycerol between lipid extracts from fibroblasts from wild-type mice or from DGKα knockout mice. However, the cells from the DGKα knockout mice had a higher concentration of DAG, consistent with the lack of downregulation of the major fraction of DAG by DGKα, in contrast with DGKε that is primarily responsible for enrichment of GPInsP_n with arachidonoyl acyl chains.