Stringency in ribosomal RNA (rRNA)-targeted fluorescence in situ hybridization (FISH) is typically adjusted with formamide, and the optimum formamide concentration at which the probe can hybridize with the target rRNA, but not with rRNAs with mismatches, is to be found experimentally. This is a difficult task when target or closest non-targets of concern. The objective of this work was to formulate mechanistic models capable of simulating the effect of formamide on probe dissociation. Using a previously described equilibrium model of FISH [Yilmaz and Noguera (2004) Applied and Environmental Microbiology 70(12):7126-7139] as the basis, the effect of formamide on free energy changes of probe-target duplex-formation (Delta G(1)(0)) and folding of target region (Delta G(3)(0)) was simulated to be linear, and models differing in the definitions of the slopes of these relationships (m(1) and m(3)) were calibrated using experimental dissociation profiles for 27 probes targeting the 16S rRNA of Escherichia coli (E. coli). A good level of predictive power was obtained when m(1) was linearly related to probe length and when m(3) was made proportional to Delta G(3)(0). The effect of single mismatches on probe dissociation with formamide was also studied, although at a preliminary level. The expected changes in Delta G(1)(0) with the introduction of mismatches were not sufficient to capture the overall trends of mismatched dissociation profiles. In conclusion, this study offers the first theoretical method to calculate dissociation profiles for perfectly matched probes, and suggests a direction to systematically evaluate the effect of formamide on mismatched probes.