The ascomycete Gibberella fujikuroi mating population C (MP-C) is well known for the production of gibberellins, but also produces many other secondary metabolites, including the red polyketide pigment bikaverin. Here, we used a differential display method to clone a polyketide synthase gene pks4 responsible for the first step of bikaverin biosynthesis. Sequence analysis indicated that pks4 encoded a 2009-amino acid polypeptide consisting of four functional domains: beta-ketoacyl synthase (KS), acyltransferase (AT), acyl carrier (ACP), and thioesterase (TE). Disruption of pks4 resulted in the loss of both pks4 transcripts and bikaverin biosynthesis in G. fujikuroi cultures. Expression of pks4 is strongly repressed by high amounts of ammonium and basic pH. Unexpectedly, pks4 was overexpressed in mutants of the regulatory gene, areA, which is responsible for the activation of nitrogen assimilation genes. Three additional polyketide synthase genes have been cloned from G. fujikuroi MP-C by heterologous hybridization. The presence of these four PKS genes demonstrates the diversity of polyketide biosynthetic pathways in this fungus.