Hypoxia-inducible factor 1 (HIF-1) is a critical transcription factor for the adaptation to lowered oxygen environments. We have previously reported that hypoxia induced phosphatidic acid (PA) accumulation through diacylglycerol kinase (DGK) activity and provided evidence that this PA production regulated HIF-1 expression. Here we report that hypoxia also produces a marked intracellular accumulation of diacylglycerol (DAG) in different cell types. The previously proposed inhibitor of phosphatidylcholine phospholipase C (PC-PLC)/sphingomyelin synthase (SMS) activities, D609, specifically abrogates both hypoxia-dependent DAG accumulation and hypoxia-induced HIF-1 expression. We show that DAG-dependent protein kinase C (PKC) isoforms do not play an essential role in the regulation of HIF-1 expression. D609 inhibits PA accumulation triggered by hypoxia, suggesting that DAG could act as substrate for its conversion into PA by DGK upon these conditions. Therefore, this work provides novel evidence for the existence of DAG/PA-dependent intracellular mechanisms involved in the regulation of HIF-1 expression. (C) 2004 Elsevier Inc. All rights reserved.