The metabolism of arachidonic acid (AA) leads to the generation of biologically active metabolites that have been implicated in cell growth and proliferation, as well as survival and apoptosis. We have previously demonstrated that rat Walker 256 (W256) carcinosarcoma cells express the platelet-type 12-lipoxygenase (12-LOX) and synthesize 12(S)- and 15(S)-HETE as their major LOX metabolites. Here we show that Walker 256 cells also express leukocyte-type 12-LOX and that its overexpression in these cells significantly extends their survival and delays apoptosis when cells are cultured under serum-free conditions. Under serum-free conditions, the expression of leukocyte-type 12-LOX is upregulated. 12-LOX-transfected W256 cells had a more spread morphology in culture compared with wild-type or mock-transfected cells. Examination of W256 cells showed that the cells expressed a number of integrins on their surface. Overexpression of 12-LOX enhanced the surface expression and focal adhesion localization of integrin alphavbeta5, while not affecting other integrins. Also, the 12-LOX-transfected W256 cells exhibited higher levels of microfilament content. Treatment of cells with monoclonal antibody to alphavbeta5 or cytochalasin B (a microfilament-disrupting agent), but not antibodies to other integrin receptors, resulted in significant apoptosis, characterized by rapid rounding up and detachment from the substratum. These results show that the 12-LOX pathway is a regulator of cell survival and apoptosis, by affecting the expression and localization of the alphavbeta5 integrin and actin microfilaments in Walker 256 cells.