Knowledge of heat-responsive proteins is critical for further understanding of the molecular mechanisms of heat tolerance. The objective of this study was to compare proteins differentially expressed in two C(3) grass species contrasting in heat tolerance, heat-tolerant thermal Agrostis scabra and heat-sensitive Agrostis stolonifera L., and to identify heat-responsive proteins for short- and long-term responses. Plants were exposed to 20/15 degrees C (day/night, control) or 40/35 degrees C (day/night, heat stress) in growth chambers. Leaves were harvested at 2 and 10 days after temperature treatment. Proteins were extracted and separated by fluorescence difference gel electrophoresis (DIGE). Thermal A. scabra had superior heat tolerance than A. stolonifera, as indicated by the maintenance of higher chlorophyll content and photochemical efficiency under heat stress. The two-dimensional difference electrophoresis detected 68 heat-responsive proteins in the two species. Thermal A. scabra had more protein spots either down- or up-regulated at 2 days of heat stress, but fewer protein spots were altered at 10 days of heat stress compared with A. stolonifera. Many protein spots exhibited transient down-regulation in thermal A. scabra (only at 2 days of heat treatment), whereas down-regulation of many proteins was also found at 10 days of heat treatment in A. stolonifera, which suggested that protein metabolism in thermal A. scabra might acclimate to heat stress more rapidly than those in A. stolonifera. The sequences of 56 differentially expressed protein spots were identified using mass spectrometry. The results suggest that the maintenance or less severe down-regulation of proteins during long-term (10 days) heat stress may contribute to the superior heat tolerance in thermal A. scabra, including those involved in photosynthesis [RuBisCo, RuBisCo activase, chloroplastic glyceraldehydes-3-phosphate dehydrogenase (GAPDH), chloroplastic aldolase, oxygen-evolving complex, photosystem I subunits], dark respiration (cytosolic GAPDH, cytoplasmic aldolase, malate dehydrogenase, hydroxypyruvate reductase, sedoheptulose-1,7-bisphosphatase), photorespiration [(hydroxypyruvate reductase, alanine aminotransferase (AlaAT), hydroxymethyltransferase (SHMT), glycine decarboxylase (GDC)], as well as heat and oxidative stress protection [heat shock cognate (HSC) 70 and FtsH-like protein].