Penicillium brocaestrain P6 is a phosphate-solubilizing fungus isolated from farmland in Guangdong Province, China. To gain better insights into the phosphate solubilization mechanisms of strain P6, a T-DNA insertion population containing approximately 4500 transformants was generated byAgrobacterium tumefaciens-mediated transformation. The transformation procedure was optimized by using a Hybond N membrane for co-cultivation ofA. tumefaciensandP. brocae. A mutant impaired in phosphate solubilization (named MT27) was obtained from the T-DNA insertion population. Thermal asymmetric interlaced PCR was then used to identify the nucleotide sequences flanking the T-DNA insertion site. The T-DNA in MT27 was inserted into the fourth exon of an enolase gene, which shows 90.8 % nucleotide identity with enolase mRNA fromAspergillus neoniger. Amino acid sequence homology analysis indicated that the enolase is well conserved among filamentous fungi andSaccharomyces cerevisiae. Complementation tests with the MT27 mutant confirmed that the enolase gene is involved in phosphate solubilization. Analysis of organic acids in culture supernatants indicated reduced levels of oxalic acid and lactic acid for the MT27 mutant compared to the parent strain P6 or the complementation strain. In conclusion, we suggest that the identified enolase gene ofP. brocaeis involved in production of specific organic acids, which, when secreted, act as phosphate solubilizing agents.