We have constructed a series of deletion mutants of Arabidopsis MAPK kinase kinase (AtMEKK1) and obtained a constitutively active mutant, AtMEKK1Delta166, which lacks in self-inhibitory sequence of N-terminal 166 amino acids but still has substrate specificity. AtMEKK1Delta166 predominantly phosphorylates AtMEK1, an Arabidopsis MAPKK, but not its double mutant (AtMEK1T218A/S224E), suggesting that Thr-218 and Ser-224 are the phosphorylation sites. In wounded seedlings, AtMEKK1 was activated and phosphorylated its downstream AtMEK1. Furthermore, analysis using anti-AtMEKK1 and anti-AtMEK1 antibodies revealed that the interaction between the two proteins was signal dependent. These results suggest the presence of AtMEKK1-AtMEK1 pathway induced by wounding.