High temperature and humidity (HTH) stress during soybean seed development and maturity in the field easily leads seed to pre-harvest deterioration. However, how proteins and their involved pathways in developing soybean seed systematically cause deterioration is still not largely understood. To reveal it, we compared the proteome composition of developing seed (R-7 period) of a pre-harvest seed deterioration sensitive soybean cultivar at different HTH stress time points (24, 96 and 168 h) with their corresponding controls by 2-DE. 42 protein spots were found to be differentially expressed and successfully identified by MALDI-TOF MS to match 31 diverse protein species. These proteins were involved in 13 cellular responses and metabolic processes including carbohydrate metabolism, signal transduction, protein biosynthesis, photosynthesis, protein folding and assembly, energy pathway, cell rescue and defense, cell cycle, nitrogen metabolism, lipid metabolism, amino acid metabolism, transcription regulation, and secondary metabolite biosynthesis. Based on these proteins' functions and involved pathways, together with ultrastructural, physical and chemical, and metabolomic data, a pre-harvest seed deterioration mechanism was proposed. Such a mechanism allows us to further understand the possible management strategy of cellular activities occurring in the HTH-stressed developing seeds and provides new insights into the HTH stress responses in crop developing seeds. (C) 2012 Elsevier B.V. All rights reserved.