Heterologous expression of Pleurotus eryngii versatile peroxidase (VP) in Escherichia coli was investigated, The cDNA encoding the mature sequence of the allelic variant VPL2 was cloned into the expression vector pFLAG1 and expressed in E. coli W3110. After induction with isopropyl-beta-D-thiogalactopyranoside (IPTG), the recombinant polypeptide (VPL2*) was found to be the major protein located in inclusion bodies. In vitro folding of VPL2* was initially performed under conditions previously determined for folding of recombinant lignin peroxidase from Phanerochaete chrysosporium (LiPH8*) but a very low yield of active enzyme was obtained (<0.1% of the total protein in the folding reaction). The influence of different parameters in VPL2* folding was investigated and the result compared with those obtained for other peroxidases. Up to 7% folding yield was achieved with VPL2* using optimised conditions which included: 0.15 M urea, 5 mM Ca2+, 20 muM haemin, a 4:1 oxidised-glutathione/reduced-glutathione ratio and 0.1 mg/ml protein concentration at pH 9.5, a yield twice as high as previously obtained for other peroxidases from Classes II or III. The enzyme presented spectral and kinetic properties identical to those of the fungally derived protein. It was fully functional in both Mn-mediated and Mn-independent peroxidase assays.