A number of recent studies have highlighted the presence of a nuclear pool of inositol lipids   that is regulated during progression through the cell cycle  , differentiation   and after DNA damage , suggesting that a number of different regulatory pathways impinge upon this pool of lipids. It has been suggested that the downstream consequence of the activation of one of these nuclear phosphoinositide (PI) regulatory pathways is the generation of nuclear diacylglycerol (DAG)   , which is important in the activation of nuclear protein kinase C (PKC)   . Activation of PKC in turn appears to regulate the progression of cells through G1 and into S phase  and through G2 to mitosis     . Although the evidence is enticing, there is as yet no direct demonstration that nuclear PIs can be hydrolysed to generate nuclear DAG. Previous data in murine erythroleukemia (MEL) cells have suggested that nuclear phosphoinositidase Cbeta1 (PIC-beta1) activity is important in the generation of nuclear DAG. Here, we demonstrate that the molecular species of nuclear DAG bears little resemblance to the PI pool and is unlikely to be generated directly by hydrolysis of these inositol lipids. Further, we show that there are in fact two distinct subnuclear pools of DAG; one that is highly disaturated and mono-unsaturated (representing more than 90% of the total nuclear DAG) and one that is highly polyunsaturated and is likely to be derived from the hydrolysis of PI. Analysis of these pools, either after differentiation or during cell-cycle progression, suggests that the pools are independently regulated, possibly by the regulation of two different nuclear phospholipase Cs (PLCs).