The tobacco NPK1 cDNA was the first-isolated plant cDNA for a homolog of mitogen-activated protein kinase kinase kinases (MAPKKKs). The kinase domain of the NPK1 protein can replace the functions of MAPKKKs in yeasts, while the amino acid sequence of the kinase-unrelated region does not have any homology to those of MAPKKKs from other organisms. Transcription of the NPK1 gene takes place in meristematic tissues or immature organs in a tobacco plant. During a tobacco cell cycle, transcriptional and translational products of NPK1 are present from S to M phase and decrease after the M phase. Expression of the NACK1 gene, which is predicted to encode a novel kinesin-like microtubule-based motor protein capable of activating NPK1, is specific to M phase, suggesting that activation of NPK1 occurs in M phase. Characterization of cDNAs for a MAPKK and a MAPK which can act downstream of NPK1 makes a proposition that the MAP kinase pathway involving NPK1 regulates a mitotic process associated with microtubules.