Abstract: In S. cerevisiae, the mating, filamentous growth (FG) and high-osmolarity glycerol (HOG) MAP kinase signaling pathways share components, yet mediate distinct responses to different extracellular signals. Cross-talk is suppressed between the mating and FG pathways because mating signaling induces the destruction of the FG transcription factor Tec1. Here we show that HOG pathway activation results in phosphorylation of the FG MAPK, Kss1, and the MAPKK, Ste7. However, FG transcription is not activated because HOG signaling prevents the activation of Tec1. In contrast to the mating pathway, we find that the mechanism involves the inhibition of DNA binding by Tec1 rather than its destruction. We also find that nuclear accumulation of Tec1 is not affected by HOG signaling. Inhibition by Hog1 is apparently indirect since it does not require any of the consensus S/TP MAPK phosphorylation sites on Tec1, its DNA binding partner Ste12, or the associated regulators Dig1 or Dig2. It also does not require the consensus MAPK sites of the Ste11 activator Ste50, in contrast to a recent proposal for a role for negative feedback in specificity. Our results demonstrate that HOG signaling interrupts FG pathway signal transduction between the phosphorylation of Kss1 and the activation of DNA binding by Tec1.