We have generated mice carrying the most common mutation in cystic fibrosis (CF), ΔF508, within the cystic fibrosis (Cftr) gene. Mutant animals show pathological and electrophysiological changes consistent with a CF phenotype. ΔF508^−/− mice die from peritonitis and show deficiencies in cAMP–activated electrogenic Cl⁻ transport. These mice produce ΔF508 transcripts and show the temperature–dependent trafficking defect first described for the human ΔF508 CFTR protein. A functional CFTR Cl⁻ channel not demonstrated by null CF mice or present at 37 °C was detected following incubation of epithelial cells at 27 °C. Thus, these mice are an accurate ΔF508 model and will be valuable for testing drugs aimed at overcoming the ΔF508 trafficking defect.