The effects of environmental chemicals have traditionally been detected by monitoring biomarkers of exposure or biomarkers of effect. Proteomics, the study of the complete profile of proteins in a given cell, tissue or biological system, is a new approach using a set of high-throughput methodologies with a wide dynamic range that makes possible the discovery of novel biomarkers. This article reviews the application of two-dimensional electrophoresis and mass-spectrometry methods to environmental toxicology. Emphasis is placed on the protein-expression signature approach and on identifying redox-based post-translational protein modifications. The methodological links between studies in sentinel organisms and humans are explored. Significant limitations and challenges are placed on this approach by the shortage of genome sequence data necessary for protein identification and the growing requirement for more stringent study design. Proteomics will continue to be an important toolkit to help address the growing environmental threat posed by nanoparticles and endocrine disrupting agents.