The hormonally regulated Ca2+-dependent enzyme, cytosolic phospholipase A(2), (cPLA(2)) is activated by a range of inflammatory stimuli. Tumour necrosis factor-alpha (TNF) is one of the first known Stimuli for cPLA, but it is not known whether both TNF receptor subtypes are involved in activating the lipase. In the present study, we show for the first time that both type 1 55 kDa TNFR (TNFR1) and type 11 75 kDa TNFR (TNFR2) stimulate cPLA(2) enzyme, but with distinct signalling mechanisms. TNFR I activates mitogen-activated protein kinase (MAPK) and p38MAPK. TNFR1 then phosphorylates and activates cPLA(2) in a MAPK-dependent fashion. Furthermore, TNFR1 causes the translocation and caspase-dependent proteolysis of cPLA, as part of its activation profile. TNFR2, on the other hand, does not cause the phosphorylation of cPLA(2) as it does not activate MAPK or p38MAPK, but instead activates cPLA(2) by causing its translocation to plasma membrane and perinuclear subcellular regions. TNFR2 activation causes a delayed, slight increase in [Ca2+](i) of < 50 nM that may contribute towards the translocation and activation of cPLA(2). Therefore both TNF receptor subtypes play a role in cPLA(2) activation, but by means of separate signal-transduction pathways.