This study evaluated the influence of four chemical pretreatment agents (HNO3, catalyzed H2O2, KMnO4, NaOH) on the subsequent enzymatic conversion of subbituminous coal by a fungal manganese peroxidase (MnP) produced by the agaric white-rot fungus Bjerkandera adusta. The effects of the combined chemical and enzymatic treatments were analyzed by high performance size exclusion chromatography (HPSEC) and 3-dimensional excitation emission matrix fluorescence spectroscopy (3D-EEM). The nature of pretreatment agents and their applied concentrations had significant impacts on subsequent enzymatic conversion of coal. The 3D-EEM spectroscopic analysis provided new insight into the nature of the depolymerized and released coal constituents. Using the fluorescence spectra, it was possible to distinguish among humic-like, fulvic acid-like, protein-like, and aromatic/PAH-like substances. The fungal enzyme MnP had little effect on the untreated coal controls. Nitric acid (HNO3) was the most effective pretreatment agent as indicated by the HPSEC profiles, followed by catalyzed H2O2 and KMnO4. Low molecular weight aromatic fragments with sizes ranging from 1.1 to 6.2 kDa were released by all of the pretreatment agents used in combination with MnP. For KMnO4 and HNO3 pretreated coal, all four EEM regions increased after MnP treatment (for example, 307.5/422 nm and 232.5/426 nm, and 340/448 nm and 242.5/484 nm for humic and fulvic acid-like fragments, respectively). (C) 2013 Elsevier Ltd. All rights reserved.