Stress-activated MAP kinases (SAPKs) respond to a wide variety of stressors. In most cases, the pathways through which specific stress signals are transmitted to the SAPKs are not known. The Saccharomyces cerevisiae SAPK Mpk1 (Slt2) is a well-characterized component of the cell-wall integrity (CWI) signaling pathway, which responds to physical and chemical challenges to the cell wall. However, Mpk1 is also activated in response to genotoxic stress through an unknown pathway. We show that, in contrast to cell-wall stress, the pathway for Mpk1 activation by genotoxic stress does not involve the stimulation of the MAP kinase kinases (MEKs) that function immediately upstream of Mpk1. Instead, DNA damage activates Mpk1 through induction of proteasomal degradation of Msg5, the dual-specificity protein phosphatase principally responsible for maintaining Mpk1 in a low-activity state in the absence of stress. Blocking Msg5 degradation in response to genotoxic stress prevented Mpk1 activation. This work raises the possibility that other Mpk1-activating stressors act intracellularly at different points along the canonical Mpk1 activation pathway.