Ozone is the most persistent, wide-spread air pollutant in the United States. Over one half of the population of the US lives in cities or suburban areas which do not meet the National Ambient Air Quality Standard for ozone which is 0.12 ppm averaged over 1 h. Controlled laboratory exposures of human subjects have shown that ozone exposure produces decreased pulmonary function, hyperresponsiveness to inhaled methacholine, inspiratory pain, and airway inflammation as assessed by bronchoalveolar lavage. However, the cellular mechanisms responsible for such effects are incompletely known. The present study examined the effects of ozone exposure at 0.50 ppm for 3 h on three types of cultured respiratory epithelial cells; primary cultures of human nasal cells and primate bronchial cells, and the A549 type II pneumocyte-derived cell line. Cells were grown to confluent monolayers in plastic 6-well plates and then exposed to ozone or filtered air on a tilting platform over a heated water bath. Lactose dehydrogenase release was significantly increased following ozone exposure of all cell types; a 75% increase from human nasal cells (P = 0.0002), a 79% increase from primate bronchial cells (P = 0.003), and a 69% increase from A549 cells (P = 0.02). These data suggest that even brief ozone exposure causes membrane injury to cultured human respiratory epithelial cells.