The α, ζ, and ε isoforms of diacylglycerol kinase exhibit a high degree of stereospecificity in the phosphorylation of diacylglycerol. In comparison, a multiple lipid kinase, MuLK, shows much less stereospecificity, phosphorylating 1,2-dioleoylglycerol only ∼2−3 times more rapidly than 2,3-dioleoylglycerol. The α and ζ isoforms of diacylglycerol kinase are inhibited by 2,3-dioleoylglycerol, but not the more substrate-selective ε isoform. The inhibition by 2,3-dioleoylglycerol is uncompetitive. This corresponds to a kinetic scheme in which the inhibitor can bind to the enzyme−substrate complex, but not to the free enzyme. Our data indicate that despite their similar structures, 1,2-dioleoylglycerol and 2,3-dioleoylglycerol do not compete for the active site of these three isoforms of diacylglycerol kinase. We suggest that the 2,3-dioleoylglycerol binds to a site on the α and ζ isoforms of diacylglycerol kinase that is exposed as a consequence of the substrate binding to the active site. The chiral specificity of these enzymes thus mimics the substrate specificity, with MuLK being the least selective and the ε isoform of diacylglycerol kinase exhibiting the greatest selectivity.