The roles of the Escherichia coli IbpA and IbpB chaperones in protection of heat-denatured proteins against irreversible aggregation in vivo were investigated. Overproduction of IbpA and IbpB resulted in stabilization of the denatured and reversibly aggregated proteins (the S fraction), which could be isolated from E. coli cells by sucrose gradient centrifugation. This finding is in agreement with the present model of the small heat-shock proteins' function, based mainly on in vitro studies. Deletion of the ibpAB operon resulted in almost twofold increase in protein aggregation and in inactivation of an enzyme (fructose-1,6-biphosphate aldolase) in cells incubated at 50 SC for 4 h, decreased efficiency of the removal of protein aggregates formed during prolonged incubation at 50 SC and affected cell viability at this temperature. IbpA/B proteins were not needed for removal of protein aggregates or for the enzyme protection/renaturation in cells heat shocked at 50 SC for 15 min. These results show that the IbpA/B proteins are required upon an extreme, long-term heat shock. Overproduction of IbpA but not IbpB caused an increase of the level of β-lactamase precursor, which was localized in the S fraction, together with the IbpA protein, which suggests that the unfolded precursor binds to IbpA but not to IbpB. Although in the wild-type cells both E. coli small heatshock proteins are known to localize in the S fraction, only 2 % of total IbpB co-localized with the aggregated proteins in the absence of IbpA, while in the absence of IbpB, the majority of IbpA was present in the aggregates fraction.