Apoptosis signal-regulating kinase 1 (ASK1) is a serine/ threonine kinase that mediates cell stress signaling initiatedby diverse stimuli, such as H2O2 andTNF a. Owing to its critical role in promoting apoptosis, ASK1 activity is highly controlledin cells. Phosphorylation of ASK1 at Thr-845 has been correlatedwith its activation, while phosphorylation at Ser-967 negatively controls its death promoting activity. Here, we report the identification of a novel phosphorylation site at Ser-1034 in the Cterminal regulatory domain of ASK1. Mutating Ser-1034 to an unphosphorylatable Ala ledto increased catalytic activity of ASK1 andenhancedproapop totic function of ASK1. Thus, the proapoptotic function of ASK1 is suppressedin part by phosphorylation at its C-terminal regulatory domain, which may couple upstream survival kinases to the death regulatory machinery.