The generation of the lipid signalling molecules, diacylglycerol (DAG) and phosphatidic acid (PA), has been implicated in the transduction events essential for proliferation of murine B6SUt.EP stem cells responsive to erythropoietin (EPO). Some of the responses were rapid and transient while others were slower and sustained. In an attempt to better understand the biphasic nature of DAG and PA appearance in response to EPO, an analysis of the molecular species of DAG, phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and PA in control and EPO-treated B6SUt.EP cells was made by HPLC and TLC. Fifteen to eighteen species were identified, which were increased non-uniformly by 0.2 unit/ml EPO. Greater increases (x6) were observed in 16:0,20:4 and 18:0,20:4 DAGs than in other species. The molecular species profiles of the stimulated DAGs were compared with the profiles of molecular species contained in the phospholipids. Comparison of the increase in DAG species caused by EPO with the molecular species present in PC and PI showed both PI and PC as the source of the fast DAG accumulation and only PC as the source of the slow DAG accumulation. PE was involved in both phases. We found a consistent formation of ethanolamine over time, in larger amounts than choline, providing strong evidence that, in addition to PC, PE is a major substrate. In addition, changes in molecular species of PA in response to EPO suggest that PI cannot account for the mass of PA formed during the first 30 s incubation with EPO, nor for PA formed during 30 min with EPO. It is concluded that the majority of PA was formed by a direct action of phospholipase D on PC.