Airway epithelial cells (AEC) contain both pro- and anti-apoptotic factors but little is known about mechanisms regulating apoptosis of these cells. In this study we have examined the localization of pro-caspase-3 and Zn2+, a cellular regulator of procaspase-3, in primary sheep and human AEC. Zn2+ was concentrated in both cytoplasmic vesicles and ciliary basal bodies, in the vicinity of both pro-caspase-3 and the antioxidant Cu/Zn superoxide dismutase (Cu/Zn SOD). Depletion of intracellular Zn2+ in sheep AEC, using the membrane permeant Zn2+ chelator TPEN, increased lipid peroxidation in the apical cell membranes (as assessed by immunofluorescence with anti-hydroxynonenal) as well as increasing activated pro-caspase-3 and apoptosis. There were smaller increases in caspase-2 and -6 but not other caspases. Activation of caspase-3 in TPEN-treated AEC was inhibited strongly by N-acetylcysteine and partially by vitamin C and vitamin E. These findings suggest that cytoplasmic pro-caspase-3 is positioned near the lumenal surface of AEC where it is under the influence of Zn2+ and other anti-oxidants. (C) 2002 Elsevier Science (USA). All rights reserved.