A stochastic Petri net model was developed for simulating the σ32 stress circuit in E. coli. Transcription factor σ32 is the principal regulator of the response of E. coli to heat shock. Stochastic Petri net (SPN) models are well suited for kinetics characterization of fluxes in biochemical pathways. Notably, there exists a one-to-one mapping of model tokens and places to molecules of particular species. Our model was validated against experiments in which ethanol (inducer of heat shock response) and σ32-targeted antisense (downward regulator) were used to perturb the σ32 regulatory pathway. The model was also extended to simulate the effects of recombinant protein production. Results show that the stress response depends heavily on the partitioning of σ32 within the cell; that is, σ32 becomes immediately available to mediate a stress response because it exists primarily in a sequestered, inactive form, complexed with chaperones DnaK, DnaJ, and GrpE. Recombinant proteins, however, also compete for chaperone proteins, particularly when folded improperly. Our simulations indicate that when the expression of recombinant protein has a low requirement for DnaK, DnaJ, and GrpE, the overall σ32 levels may drop, but the level of heat shock proteins will increase. Conversely, when the overexpressed recombinant protein has a strong requirement for the chaperones, a severe response is predicted. Interestingly, both cases were observed experimentally. © 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 75: 120–129, 2001.