Transcription promoters of heat shock protein (HSP) genes have been used to control the expression of heterologous proteins in plants and plant cells. To obtain a strong HSP promoter that is functionally active in Nicotiana tabacum BY-2 cells, we set out to identify a promoter of an endogenous gene showing high activation of expression by heat. An N. tabacum BY-2 cell culture was treated for 8 h at 37°C and the cell protein extract analyzed by two-dimensional electrophoresis. A major spot was identified by mass spectrometry as belonging to the small HSP family. The promoter regions and the 5' and 3' untranslated regions of two genes, NtHSP3A and NtHSP3B, with sequences fitting the protein identified were cloned and fused to a hybrid reporter gene coding for β-glucuronidase (GUS) and a yellow fluorescent protein. These constructs were introduced into N. tabacum BY2 cells by Agrobacterium tumefaciens-mediated transformation. Both promoters conferred similar heat-induced GUS expression. In the best heat shock condition, GUS activity was increased 200 fold and reached 285 pmol min(-1) μg protein(-1). Up-scaling in a 4-l bioreactor resulted in similar heat-induced expression. The NtHSP3A promoter was then used to drive the expression of NtPDR1, a plasma membrane transporter belonging to the pleiotropic drug resistance family. No expression was observed at 25°C, while, at 37°C, expression was similar to that obtained using a strong constitutive promoter. These data show that the HSP promoters isolated are useful for high heat-induced expression in N. tabacum BY-2 cells.