Two genes encoding MAP kinase homologs, designated as mpkB and mpkC, were isolated from Aspergillus nidulans by PCR with degenerate primers. Deletion and over-expression mutants of mpkC showed no detectable phenotypes under any external stress tested. Deletion of mpkB caused pleiotropic phenotypes including a failure in forming cleistothecia under any induction conditions for sexual development, increased Hulle cell production, slow hyphal growth and aberrant conidiophore morphology. Over-expression of mpkB led to increased cleistothecium production. While the transcripts of mpkB and mpkC were constitutively synthesized through the entire life cycle, their size and amount differed with developmental stages. An outcross test using fluorescent protein reporters showed that the mpkB deletion mutant could not form heterokaryons with wild type. Protoplast fusion experiments showed that the fusant of the mpkB mutant with wild type could undergo normal sexual development. However, heterokaryotic mycelia that were produced from a fusant between two mpkB deletion mutants could not form cleistothecia, although they did appear to form diploid nuclei. These results suggest that the MpkB MAP kinase is required for some post-karyogamy process as well as at the hyphal anastomosis stage to accomplish sexual development successfully.