The diacylglycerol kinase from E. coli transfers some of the γ-phosphate of ATP to water as well as to diacylglycerol. We also demonstrate that glycerol can act as an acceptor for the phosphate of ATP. We have compared this behavior with that of the only mammalian isoform of diacylglycerol kinase that exhibits acyl chain specificity, i.e. DGKɛ. The purpose of the study was to determine if differences in the competition between ATPase activity and lipid phosphorylation could contribute to the observed acyl chain specificity with different diacylglycerols. Neither with the highly specific substrate of DGKɛ, 1-stearoyl-2-arachidonoyl glycerol, nor with a less specific substrate, 1-stearoyl-2-linoleoyl glycerol, is there any evidence for ATP hydrolysis accompanying substrate phosphorylation. Thus, at least for this isoform of diacylglycerol kinase, water does not compete with diacylglycerol as an acceptor of the γ-phosphate of ATP. The results demonstrate that the substrate specificity of mammalian DGKɛ is not a consequence of different degrees of ATP hydrolysis in the presence of different species of diacylglycerol.