Horseradish peroxidase is used in many biotechnological fields including diagnostics, biocatalysts and biosensors. Horseradish peroxidase isozyme C (HRPC) was extracellularly expressed in Spodoptera frugiperda Sf9 cell culture and in intact larvae. At day 6 post-infection, the concentration of active HRPC in suspension cultures was 3.0 +/- 0.1 mu g per 1 x 10(6) cells or 3.0 +/- 0.1 mg l(-1) with a multiplicity of infection of 1 in the presence of 7.2 mu M hemin. Similar yields were obtained in monolayer cultures. In larvae, the HRPC expression level was 137 +/- 17 mg HRPC kg(-1) larvae at day 6 post-infection with a single larvae thus producing approximately 41 mu g HRPC. The whole larval extract was separated by ion exchange chromatography and HRPC was purified in a single step with a yield of 75% and a purification factor of 117. The molecular weight of recombinant HRPC was 44,016 Da, and its glycosylation pattern agreed with that expected for invertebrates. The K-m and V-max were 12.1 +/- 1.7 mM and 2673 +/- 113 U mg(-1), respectively, similar to those of HRP purified from Annoracia rusticana roots. The method described in this study, based on overexpression of HRPC in S. frugiperda larvae, is a simple and inexpensive way to obtain high levels of active enzyme for research and other biotechnological applications. (C) 2010 Elsevier Ltd. All rights reserved.