To explore the impact of a chloride gradient on the measurement of CFTR activity in an Ussing chamber, donor‐matched non‐CF nasal epithelia, generated from expanded primary cells, were assayed in either symmetrical chloride solutions (referred to as a “0% gradient”) or with an imposed basolateral to apical chloride gradient (50 or 100%). This evidence concerns the gene CFTR and cystic fibrosis.