The metabolism of pyrene by Penicillium glabrum strain TW 9424, a strain isolated from a site contaminated with polycyclic aromatic hydrocarbons (PAHs) was investigated in submerged cultures. The metabolites formed were identified as 1-hydroxypyrene, 1,6- and 1, 8-dihydroxypyrene, 1,6- and 1,8-pyrenequinone, and 1-pyrenyl sulfate. In addition, two new metabolites were isolated and identified by UV, 1H nuclear magnetic resonance, and mass spectroscopy as 1-methoxypyrene and 1,6-dimethoxypyrene. Experiments with [methyl-3H]S-adenosyl-l-methionine (SAM) revealed that SAM is the coenzyme that provides the methyl group for the methyltransferase involved. To our knowledge, this is the first time that methoxylated metabolites of PAHs have been isolated from fungal cultures.