Summary Arabidopsis ROF1 (AtFKBP62) is a peptidyl prolyl cis / trans isomerase and a member of the FKBP (FK506 binding protein) family. ROF1 expression is induced by heat stress and developmentally regulated. In this study, we show that ROF1 binds heat shock proteins HSP90.1 via its tetratricopeptide repeat domain, and localizes in the cytoplasm under normal conditions. Exposure to heat stress induces nuclear localization of the ROF1–HSP90.1 complex, which is dependent upon the presence of the transcription factor HsfA2, which interacts with HSP90.1 but not with ROF1. Nuclear localization of ROF1 was not detected in Arabidopsis HSP90.1 and HsfA2 knockout mutants. The rof1 knockout plants exhibited collapse when 24–48 h passed between acclimation at 37°C and exposure to 45°C. Transgenic ROF1 over‐expressors showed better survival in response to exposure to 45°C than wild‐type plants did. In rof1 knockout mutants, the level of expression of small HSPs regulated by HsfA2 was dramatically reduced after exposure to 37°C and recovery for 24–48 h, and correlates well with the mutant phenotype. We suggest a role for ROF1 in prolongation of thermotolerance by sustaining the levels of small HSPs that are essential for survival at high temperatures.