Aims: Purification and characterization of the high molecular mass Candida albicans-killing protein secreted by Penicillium chrysogenum. Methods and Results: The protein was purified by a combination of ultrafiltration, chromatofocusing and gel filtration. Enzymological characteristics [relative molecular mass (M-r) = 155 000, subunit structure alpha(2) with M-r,M-alpha = 76 000, isoelectric point (pI) = 5.4] were determined using SDS-PAGE and 2D-electrophoresis. N-terminal amino acid sequencing and homology search demonstrated that the antifungal protein was the glucose oxidase (GOX) of the fungus. The enzyme was cytotoxic for a series of bacteria, yeasts and filamentous fungi. Vitamin C (1.0 mg ml(-1)) prevented oxidative cell injuries triggered by 0.004 U GOX in Emericella nidulans cultures but bovine liver catalase was ineffective even at a GOX : catalase activity ratio of 0.004 : 200 U. A secondary inhibition of growth in E. nidulans cultures by the oxygen-depleting GOX - catalase system was likely to replace the primary inhibition exerted by H2O2. Conclusions: Penicillium chrysogenum GOX possesses similar enzymological features to those described earlier for other Penicillium GOXs. Its cytotoxicity was dependent on the inherent antioxidant potential of the test micro-organisms. Significance and Impact of the Study: Penicillium chrysogenum GOX may find future applications in glucose biosensor production, the disinfection of medical implants or in the food industry as an antimicrobial and/or preservative agent.