The chaperonin GroEL assists protein folding in the presence of ATP and magnesium through substrate protein capsulation in combination with the cofactor GroES. Recent studies have revealed the details of folding cycles of GroEL from Escherichia coli, yet little is known about the GroEL-assisted protein folding mechanisms in other bacterial species. Using three model enzyme assays, we have found that GroEL1 from Chlamydophila pneumoniae, an obligate human pathogen, has a broader selectivity for nucleotides in the refolding reaction. To elucidate structural factors involved in such nucleotide selectivity, GroEL chimeras were constructed by exchanging apical, intermediate, and equatorial domains between E. coli GroEL and C. pneumoniae GroEL1. In vitro folding assays using chimeras revealed that the intermediate domain is the major contributor to the nucleotide selectivity of C. pneumoniae GroEL1. Additional site-directed mutation experiments led to the identification of Gln(400) and Ile(404) in the intermediate domain of C. pneumoniae GroEL1 as residues that play a key role in defining the nucleotide selectivity of the protein refolding reaction.