Escherichia coli Hsp100 ClpB plays critical roles in multi-chaperone systems in cell physiology. After being activated by protein or peptide binding, ClpB disaggregates denatured polypeptides by employing ATP hydrolysis and allows other molecular chaperones such as Hsp70 DnaK and Hsp40 DnaJ to refold the non-native polypeptides. ClpB contains two nucleotide-binding domains with Walker A and B motifs within their primary sequences. Therefore, ClpB can be classified as a member of the large ATPase family known as ATPases associated with various cellular activities (AAAs). The mechanisms by which the ClpB acts as a molecular chaperone to disaggregate denatured polypeptides are unknown. To investigate how the nucleotide-binding domain participates in ClpB chaperone activity, we have cloned and crystallized ClpB nucleotide-binding domain 1 (NBD1). The ClpB NBD1 crystals diffract to 1.80 Å using a synchrotron X-ray source and belong to the space group P212121, with unit-cell parameters a = 38.41, b = 65.48, c = 79.13 Å. Structure determination by the MAD method is under way.