The role of small heat-shock proteins in Escherichia coli is still enigmatic. We show here that the small heatshock protein IbpB is a molecular chaperone that assists the refolding of denatured proteins in the presence of other chaperones. IbpB oligomers bind and stabilize heat-denatured malate dehydrogenase (MDH) and ureadenatured lactate dehydrogenase and thus prevent the irreversible aggregation of these proteins during stress. While IbpB-stabilized proteins alone do not refold spontaneously, they are specifically delivered to the DnaK/ DnaJ/GrpE (KJE) chaperone system where they refold in a strict ATPase-dependent manner. Although GroEL/ GroES (LS) chaperonins do not interact directly with IbpB-released proteins, LS accelerate the rate of KJEmediated refolding of IbpB-released MDH, and to a lesser extent lactate dehydrogenase, by rapidly processing KJE-released early intermediates. Kinetic and gelfiltration analysis showed that denatured MDH preferentially transfers from IbpB to KJE, then from KJE to LS, and then forms a active enzyme. IbpB thus stabilizes aggregation-prone folding intermediates during stress and, as an integral part of a cooperative multichaperone network, is involved in the active refolding of stressdenatured proteins.The small heat-shock proteins (sHSPs) 1 belong to a ubiquitous family of low molecular mass (15-30 kDa), stress-induced proteins in prokaryotes and eukaryotes. Whereas various sHSPs share weak sequence homologies (1, 2), many sHSPs appear to be functionally and structurally related. Many sHSPs assemble into large globular complexes, whose oligomeric structures may vary depending on the degree of subunit phosphorylation or the concentration of ions (3-5). The overexpression of sHSPs in plant, yeast, and in mammal cells correlates with increased levels of thermal resistance (6 -9). In vitro, sHSPs specifically recognize, bind, and prevent the aggregation of non-native proteins during stress (3-5, 10), suggesting that similarly to GroEL/GroES (LS) and Hsp70 (11, 12), sHSPs can serve as an efficient binding reservoir for unstable proteinfolding intermediates during stress. However, at variance with other molecular chaperones, such as Hsp70, Hsp60, Hsp104, and Hsp90 (for a review, see Ref. 13), small HSPs do not hydrolyze ATP and do not display a specific ability to promote the correct refolding of the bound stabilized proteins (3-5).The small heat shock proteins IbpA and IbpB from Escherichia coli are two sequence-related 14-and 16-kDa proteins, respectively, co-transcribed during stress by the bacterial heatshock transcription factor 32 (14). IbpA and IbpB share a low sequence homology in their C-terminal region with sHSPs from yeast, plants, and mammals, including ␣B-crystallines (14 -19, 2). Furthermore, they seem to be distantly related to other bacterial chaperones such as PapD and Caf1M. Based on the resolved x-ray structure of the PapD chaperone and on sequence homology, a model has been proposed where the threedimensional structure of IbpB resembles that of immunoglobulin...