Platelet-activating factor (PAF) is an important mediator of cell loss following diverse pathophysiological challenges, but the manner in which PAF transduces death is not clear. Both PAF receptor-dependent and -independent pathways are implicated. In this study, we show that extracellular PAF can be internalized through PAF receptor-independent mechanisms and can initiate caspase-3-dependent apoptosis when cytosolic concentrations are elevated by approximately 15 pM/cell for 60 min. Reducing cytosolic PAF to less than 10 pM/cell terminates apoptotic signaling. By pharmacological inhibition of PAF acetylhydrolase I and II (PAF-AH) activity and down-regulation of PAF-AH I catalytic subunits by RNA interference, we show that the PAF receptor-independent death pathway is regulated by PAF-AH I and, to a lesser extent, by PAF-AH II. Moreover, the anti-apoptotic actions of PAF-AH I are subunit-specific. PAF-AH I alpha1 regulates intracellular PAF concentrations under normal physiological conditions, but expression is not sufficient to reduce an acute rise in intracellular PAF levels. PAF-AH I alpha2 expression is induced when cells are deprived of serum or exposed to apoptogenic PAF concentrations limiting the duration of pathological cytosolic PAF accumulation. To block PAF receptor-independent death pathway, we screened a panel of PAF antagonists (CV-3988, CV-6209, BN 52021, and FR 49175). BN 52021 and FR 49175 accelerated PAF hydrolysis and inhibited PAF-mediated caspase 3 activation. Both antagonists act indirectly to promote PAF-AH I alpha2 homodimer activity by reducing PAF-AH I alpha1 expression. These findings identify PAF-AH I alpha2 as a potent anti-apoptotic protein and describe a new means of pharmacologically targeting PAF-AH I to inhibit PAF-mediated cell death.