At present, there is little information on the optimization of the degradation of polycyclic aromatic hydrocarbons (PAH) by deuteromycete filamentous fungi, a reaction catalyzed by cytochrome P450 monooxygenases. We utilized response-surface methodology to determine the optimal growth conditions for the oxidation of the PAH pyrene by Penicillium janthinellum SFU403, with respect to the variables glucose concentration, nitrate concentration and bioconversion time. Models were derived for the relationship between the variables tested and the level of the pyrene oxidation products, 1-pyrenol (1-PY) and pyrenequinones (PQ). Production of 1-PY and PQ were optimized by the same glucose and nitrate concentrations: 2.5% glucose and 1.5% sodium nitrate. The optimized I-PY and PQ bioconversion times were 71 h and 73 h respectively. These conditions improved the yield of 1-PY by fivefold and PQ were more than 100-fold higher than the baseline levels obtained in this study. The optimized PQ yield represented 95% of the initial pyrene, thus the total optimised pyrene bioconversion to 1-PY and PQ was approximately 100%. Concentrations of glucose exceeding 4.0% repressed pyrene hydroxylation. Pyrene hydroxylation occurred almost exclusively during the deceleration phase of culture growth.