Patients with cystic fibrosis demonstrate a characteristic defect in epithelial chloride movement which can be demonstrated in vivo by the nasal potential difference technique. Following amiloride pre-treatment, the cystic fibrosis airway exhibits only a transient response to perfusion with low chloride solution, contrasting with the sustained hyperpolarisation seen in controls. This study further investigated the response to low chloride solution in the cystic fibrosis airway, examining the interaction between surface divalent ions and the low chloride response. Sequential perfusion with amiloride, low chloride and isoproterenol was tested in groups of cystic fibrosis subjects, with the diluent containing different concentrations of calcium and magnesium, on different days. When the low chloride response was measured with the nominally calcium-free diluents, the cystic fibrosis subjects had mean (SEM) responses of 8.0 (0.7), 8.6 (2.4) and 9.6 (1.6) mV in the presence of 0, 1, and 3 mM Mg²⁺ respectively, significantly different from the response in the presence of divalent ions. However, the subsequent response to isoproterenol was not different in the presence or absence of divalent ions. We hypothesise that perfusion of the cystic fibrosis airway with nominally calcium-free solutions reduces tonic inhibition of chloride secretion.