Southern blot analysis of genomic DNAs of several fungi that produce polyketide compounds with the 6-methylsalicylic acid synthase (MSAS) gene of Penicillium patulum as a probe indicated the presence of an MSAS-homologous gene in the (+)-geodin-producing strain IMI 16,043 of Aspergillus terreus. The gene, designated atX was cloned from an A. terreus genomic DNA library and 7588 bp of the gene together with its flanking regions were sequenced to reveal the presence of a 5.5 kb open reading frame coding for a protein of 1800 amino acids with 190 kDa molecular mass. The presence of a short (70 bp) intron near the N-terminus of the atX gene was predicted that contains the canonical GT and AG dinucleotides at its 5'- and 3'-splicing junctions. The predicted ATX polypeptide showed high homology with P. patulum MSAS along the whole sequence. On the other hand, slight homology was detected only around the beta-ketoacyl synthase regions of Aspergillus nidulans wA, PKSST and Colletotrichum lagenarium PKS1. No transcription of atX was observed throughout the culture period by Northern blotting analysis. To identify the function of the polypeptide encoded by the atX gene, its coding region was introduced into the fungal expression vector pTAex3 under the control of the amyB promoter. The constructed expression plasmid was introduced into A. nidulans. The transformant produced significant amounts of 6-methylsalicylic acid, the structure of which was identified by physicochemical analysis. This result unambiguously demonstrated that the atX gene codes for MSAS of A. terreus.