The Ussing chamber technique has contributed significantly to our understanding of the role of ion transport in the pathogenesis of human diseases like cystic fibrosis (CF). Here, we summarize protocols developed to study the Cl- channel function of the cystic fibrosis transmembrane conductance regulator (CFTR) protein in rectal biopsies from normal individuals and CF patients. These protocols can be applied to study the function and pharmacological modulation of wild-type and mutant CFTR in the context of the native epithelium. Together with sweat testing and genetic analyses, these functional measurements may aid in establishing a diagnosis of CF.