Three types of Arabidopsis cDNA (cANP1, cANP2 and cANP3) have been isolated that encode putative protein kinases, designated ANP1, ANP2 and ANP3. These kinases exhibit a high degree of homology to NPK1, a tobacco protein that is a member of the family of mitogen-activated protein kinase kinase kinases (MAPKKKs), which appears to function in the proliferation of tobacco cells. The predicted amino acid sequences of the kinase domains in the amino-terminal halves of the ANPs were more than 80% identical to that of NPK1, while the kinase-unrelated regions in the carboxy-terminal halves exhibited relatively low homology. Two species of cANP1 were identified, ANP1L cDNA (cANP1L) and ANP1S cDNA (cANP1S), which were derived from a single ANP1 gene: the former had an intron-like sequence in the coding region for the kinase-unrelated region, while the latter did not include such an intron-like sequence. cANP1L encoded a putative protein with both kinase and kinase-unrelated domains, resembling NPK1, whereas cANP1S encoded only the amino-terminal kinase domain because the intron-like sequence was absent, with resulting elimination of most of the kinase-unrelated region. Genetic analysis with mutant yeast cells showed that over-expression of cANP1L or of cANP1S activated the mating pheromone-responsive signal pathway which is mediated by a MAP kinase cascade. Moreover, the extent of such activation by cANP1S was greater than that by cANP1L. These results predict that differential splicing of the intron-like sequence in the ANP1 transcript might be at least one of the molecular mechanisms involved in the generation of active ANP1 protein kinase.