The lethal genetic disease cystic fibrosis is caused predominantly by in-frame deletion of phenylalanine 508 in the cystic fibrosis transmembrane conductance regulator (CFTR). F508 is located in the first nucleotide-binding domain (NBD1) of CFTR, which functions as an ATP-gated chloride channel on the cell surface. The F508del mutation blocks CFTR export to the surface due to aberrant retention in the endoplasmic reticulum. While it was assumed that F508del interferes with NBD1 folding, biophysical studies of purified NBD1 have given conflicting results concerning the mutation's influence on domain folding and stability. We have conducted isothermal (this paper) and thermal (accompanying paper) denaturation studies of human NBD1 using a variety of biophysical techniques, including simultaneous circular dichroism, intrinsic fluorescence, and static light-scattering measurements. These studies show that, in the absence of ATP, NBD1 unfolds via two sequential conformational transitions. The first, which is strongly influenced by F508del, involves partial unfolding and leads to aggregation accompanied by an increase in tryptophan fluorescence. The second, which is not significantly influenced by F508del, involves full unfolding of NBD1. Mg-ATP binding delays the first transition, thereby offsetting the effect of F508del on domain stability. Evidence suggests that the initial partial unfolding transition is partially responsible for the poor in vitro solubility of human NBD1. Second-site mutations that increase the solubility of isolated F508del-NBD1 in vitro and suppress the trafficking defect of intact F508del-CFTR in vivo also stabilize the protein against this transition, supporting the hypothesize that it is responsible for the pathological trafficking of F508del-CFTR.