A gene that encodes for a polyketide synthase (PKS) was cloned from the fungus Glarea lozoyensis and characterized. The gene (pks2) consists of four exons interrupted by three introns of 51, 59, and 65 bp, which are clustered at the 5' end. Its predicted product is a 1791-amino-acid protein containing five catalytic motifs typical of fungal PKSs, including a beta-ketosynthase, an acyltransferase, a dehydratase, a beta-ketoacyl reductase, and an acyl carrier region. The gene is transcribed from an initiation site located 375 bp upstream of the translational start codon and extends to a transcriptional termination site 244 bp downstream of the translational stop codon. The gene function is not required for either vegetative growth of G. lozoyensis or for production of pneumocandin, as shown by Agrobacterium-mediated pks2 gene disruption. Previously reported cluster analysis of ketosynthase motifs from 37 fungal polyketide synthases had grouped the Pks2p from G. lozoyensis with PKSs involved in the biosynthesis of 6-methylsalicylic acid. To verify the function of the gene, it was transferred into Aspergillus nidulans under the control of the trpC promoter. 5'-and 3'-RACE experiments confirmed that it was transcribed in the heterologous host, and was associated with the synthesis of a compound identified as 6-methylsalicylic acid by NMR and mass spectrometry. In G. lozoyensis, pks2 is flanked by a gene that encodes a putative drug resistance efflux pump. The Aspergillus pks2 transformants, which were arginine prototrophs, also exhibited precocious pigmentation and accumulated a benzophenone that appeared to be a precursor of emericellin (variecoxanthone B), a known product of A. nidulans. The buildup of the benzophenone may be related to the use of an alternative splice site for the removal of intron 1 of the pks2 transcript in the heterologous host.