Phosphorescence of single protein molecules has been directly observed by laser confocal microscopy. The native horseradish peroxidase (HRP) has been converted into a phosphorescent analog (Pt-HRP) by replacing the heme prosthetic group with platinum mesoporphyrin IX (Pt-MP). Oxygen quenching of the triplet state emission of single Pt-HRP molecules has been directly detected and the oxygen quenching constant could be estimated at the single molecule level. The method lays a foundation for experimental studies of the pathways of oxygen diffusion in proteins. (C) 2004 Elsevier B.V. All rights reserved.