The stimulation of cells by hormones, growth factors and neurotransmitters frequently leads to an increase in diacylglycerol concentration. This increase has often been observed to be biphasic, suggesting that the lipid messenger is produced from more than one source. Structural analysis of the diacylglycerol formed in cells has demonstrated that the initial phase consists of polyunsaturated diacylglycerol species which are predominately derived from inositol lipid hydrolysis. The later phase contains an increase in polyunsaturated, monounsaturated and saturated diacylglycerol species. Many of these species are apparently derived from phosphatidylcholine hydrolysis. Diacylglycerol is not just generated by phospholipase C action, it is also generated as a result of a phosphatidate phosphohydrolase catalysed conversion of the phosphatidate formed by phospholipase D catalysed hydrolysis of phosphatidylcholine. Species analysis of the generated phosphatidate has demonstrated the acyl chains are predominantly saturated or mono-unsaturated. Therefore, the diacylglycerol generated through this pathway will be saturated or mono-unsaturated rather than polyunsaturated. It is probable that it is only the polyunsaturated diacylglycerols which are messenger molecules and that messenger phosphatidates are saturated or mono-unsaturated. Diacylglycerol is removed from cells by a number of pathways. Of major importance is the diacylglycerol kinase catalysed pathway. Cells contain a number of isoforms of diacylglycerol kinase, of particular interest is the identification of an isoform which is specific for polyunsaturated diacylglycerols. This enzyme has been purified from the membranes of porcine testes and demonstrates an absolute specificity for polyunsaturated species. The results will be discussed in relation to the differing roles of different forms of phosphatidate and diacylglycerol as messenger molecules and the regulation of the pathways of their removal.