NOTE: THE SPECIAL CHARACTERS IN THIS ABSTRACT CANNOT BE DISPLAYED CORRECTLY ON THIS PAGE. PLEASE REFER TO THE ABSTRACT ON THE PUBLISHER’S WEBSITE FOR AN ACCURATE DISPLAY. Lindsey A. Allan1, Nick Morrice2, Suzanne Brady1, Gareth Magee1, Shalini Pathak1 & Paul R. Clarke1. Many pro-apoptotic signals activate caspase-9, an initiator protease that activates caspase-3 and downstream caspases to initiate cellular destruction1. However, survival signals can impinge on this pathway and suppress apoptosis. Activation of the Ras–Raf–MEK–ERK mitogen-activated protein kinase (MAPK) pathway is associated with protection of cells from apoptosis and inhibition of caspase-3 activation2, 3, 4, 5, although the targets are unknown. Here, we show that the ERK MAPK pathway inhibits caspase-9 activity by direct phosphorylation. In mammalian cell extracts, cytochrome c-induced activation of caspases-9 and -3 requires okadaic-acid-sensitive protein phosphatase activity. The opposing protein kinase activity is overcome by treatment with the broad-specificity kinase inhibitor staurosporine or with inhibitors of MEK1/2. Caspase-9 is phosphorylated at Thr 125, a conserved MAPK consensus site targeted by ERK2 in vitro, in a MEK-dependent manner in cells stimulated with epidermal growth factor (EGF) or 12-O-tetradecanoylphorbol-13-acetate (TPA). Phosphorylation at Thr 125 is sufficient to block caspase-9 processing and subsequent caspase-3 activation. We suggest that phosphorylation and inhibition of caspase-9 by ERK promotes cell survival during development and tissue homeostasis. This mechanism may also contribute to tumorigenesis when the ERK MAPK pathway is constitutively activated.