In the present study, we studied the involvement of A2 phospholipases (PLA2) in alpha2-adrenergic receptor-control of preadipocyte actin cytoskeleton. For that, various PLA2 inhibitors were tested on the ability of the selective alpha2-adrenergic agonist UK14304 to induce the spreading in alpha2AF2 preadipocytes. We observed that, whereas several Ca(2+)-dependent PLA2 blockers were ineffective, the Ca(2+)-independent phospholipase A2 (iPLA2) inhibitor, broenolactone (BEL), specifically blocked alpha2-adrenergic-dependent preadipocyte spreading without affecting the spreading activity of lysophosphatidic acid (LPA) or serum. BEL inhibition was completely restored by lysophosphatidic acid, but not by arachidonic acid or other fatty acids. The presence of the lysophospholipase (phospholipase B) suppressed the effect of LPA on preadipocyte spreading, but had no influence on alpha2-adrenergic-induced spreading. Thus, the extracellular production of LPA or fatty acids is not involved in iPLA2-dependent preadipocyte spreading. iPLA2 protein was found in preadipocytes but, conversely to cPLA2, did not exhibit any modification of its electrophoretic mobility after alpha2-adrenergic stimulation. We concluded that iPLA2 is involved in alpha2-adrenergic control of preadipocyte actin cytoskeleton.