Laccase produced by Coriolus hirsutus was purified to electrophoretic homogeneity by acetone precipitation, DEAE Sepharose CL-6B, Sephacryl S-200 HR, Hitrap SP, and Mono S chromatography. The purification was 14.5-fold with an overall yield of 32.3%. The enzyme is a monomeric glycoprotein with 11% carbohydrate content, an isoelectric point of 7.4, and a molecular mass of 73 kDa. The N-terminal amino acid sequence showed low homology to those of the laccases of other white-rot basidiomycetes. Spectroscopic analyses revealed a typical laccase active site in the C. hirsutus enzyme, as all three Cu centers were identified. The absorption spectrum showed a type 1 signal at around 600 nm and a type 3 signal near 330 nm. Type 3 Cu showed fluorescence emission near 418 nm and an excitation maximum at 332 nm. The EPR spectrum yielded parameters for the type 1 and type 2 Cu of g I I = 2.191 and A I I = 0.0097 cm - 1 , and g I I = 2.222 and A I I = 0.0198 cm - 1 , respectively. The highest rate of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) oxidation for the enzyme was reached at 45 o C, and the pH optima of the enzyme varied and was substrate dependent in the range of 2.5 to 4.0. The enzyme oxidized a variety of the usual laccase substrates, including lignin-related phenols and had highest affinity toward guaiacol. Under standard assay conditions, the apparent K m value of the enzyme toward guaiacol was 10.9 μM. The enzyme catalyzed single electron transfer via the phenoxy radical as an intermediate and was completely inhibited by l-cysteine and sodium azide but not by EDTA.