Biodegradation of pyrene by Mycobacterium frederiksbergense was studied in a two-phase partitioning bioreactor (TPPB) using silicone oil as non-aqueous phase liquid (NAPL). The TPPB achieved complete biodegradation of pyrene; and during the active degradation phase, utilization rates of 270, 230, 139, 82 mg l(-1)d(-1) for initial pyrene loading concentrations (in NAPL) of 1000, 600, 400 and 200 mg l(-1), respectively, were obtained. The degradation rates achieved using M. frederiksbergense in TPPB were much higher than the literature reported values for an ex situ PAH biodegradation system operated using single and pure microbial species. The degradation data was fitted to simple Monod, logistic, logarithmic, three-half-order kinetic models. Among these models, only exponential growth form of the three-half-order kinetic model provided the best fit to the entire degradation profiles with coefficient of determination (R2) value >0.99. From the experimental findings, uptake of pyrene by the microorganism in TPPB was proposed to be a non-interfacial based mechanism.