The chemokine receptor CCR5 plays an important role as an entry gate for the human immunodeficiency virus‐1 (HIV‐1) and for viral postentry events. Among signal transducers used by chemoattrac‐tant receptors' the phosphatidylcholine‐specific phos‐pholipase D (PLD) produces large amounts of second messengers in most cell types. However' the relevance of PLD isoforms to CCR5 signaling and HIV‐1 infection process remains unexplored. We show here that CCR5 activation by MIP‐1β in HeLa‐MAGI cells triggered a rapid and substantial PLD activity' as assessed by mass choline production. This activity required the activation of ERK1/2‐MAP kinases and involved both PLD1 and PLD2. MIP‐1β also promoted the activation of an HIV‐1 long terminal repeat (LTR) by the transac‐tivator Tat in HeLa P4.2 cells through a process involving ERK1/2. Expression of wild‐type and catalytically inactive PLDs dramatically boosted and inhibited the LTR activation ‘respectively’ without altering Tat expression. Wild‐type and inactive PLDs also respectively potentiated and inhibited HIV‐1 BAL replication in MAGI cells. Finally' in monocytic THP‐1 cells' antisense oligo‐nucleotides to both PLDs dramatically inhibited the HIV‐1 replication. Thus' PLD is activated downstream of ERK1/2 upon CCR5 activation and plays a major role in promoting HIV‐1 LTR transactivation and virus replication' which may open novel perspectives to anti‐HIV‐1 strategies.—Paruch S., Heinis M., Lemay J., Hoeffel G., Marañón C., Hosmalin A., Perianal A. CCR5 signaling through phospholipase D involves p44/42 MAP‐kinases and promotes HIV‐1 LTR‐directed gene expression. FASEB J . 21, 4038–4046 (2007)