Stress-activated MAPKs (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. In this study, we delineate the intracellular signaling pathway by which the trivalent toxic metalloid arsenite [As(III)] activates the yeast SAPK Hog1. We demonstrate that, to activate Hog1, As(III) must enter the cell through the glycerol channel Fps1 and must be metabolized to methyl arsenite [MAs(III)] by the dimeric methyltransferase Mtq2:Trm112. We found that Mtq2:Trm1 displays SAM-dependent methyltransferase activity toward both As(III) and MAs(III). Additionally, we present genetic and biochemical evidence that MAs(III), but not As(III), is a potent inhibitor of the protein tyrosine phosphatases (Ptp2 and Ptp3) that normally maintain Hog1 in an inactive state. Inhibition of Ptp2 and Ptp3 by MAs(III) results in elevated Hog1 phosphorylation without activation of the protein kinases that act upstream of the SAPK and raises the possibility that other Hog1-activating stressors act intracellularly at different points along the canonical Hog1 activation pathway. Finally, we show that arsenate [As(V)], a pentavalent form of arsenic, also activates Hog1, but through a pathway that is distinct from that of As(III) and involves activation of the Hog1 MEK Pbs2.