The plant hormone auxin activates many early response genes that are thought to be responsible for diverse aspects of plant growth and development. It has been proposed that auxin signal transduction is mediated by a conserved signalling cascade consisting of three protein kinases: the mitogen-activated protein kinase (MAPK), MAPK kinase (MAPKK) and MAPKK kinase (MAPKKK). Here we show that a specific plant MAPKKK, NPK1, activates a MAPK cascade that leads to the suppression of early auxin response gene transcription. A mutation in the kinase domain abolishes NPK1 activity, and the presence of the carboxy-terminal domain diminishes the kinase activity. Moreover, the effects of NPK1 on the activation of a MAPK and the repression of early auxin response gene transcription are specifically eliminated by a MAPK phosphatase. Transgenic tobacco plants overexpressing the NPK1 kinase domain produced seeds defective in embryo and endosperm development. These results suggest that auxin sensitivity may be balanced by antagonistic signalling pathways that use a distinct MAPK cascade in higher plants.