Previous studies have shown that ozone in low concentration in vivo depresses the activity of alveolar macrophage acid phosphatase, lysozyme, and β-glucuronidase. In the present studies, rabbit alveolar macrophages were held in tissue culture as a monolayer and were exposed to various concentrations of ozone when covered by only a film of tissue culture media. This approach eliminated recovery of a mixed population of cells from the lung and possible influx of macrophages in response to the noxious agent. Ozone in moderately low concentration, 2 ppm, depressed the activity of macrophage lysosomal acid hydrolases. There was slight accumulation of enzyme in extracellular media during and after three hour exposure to ozone. Hydrolase depression effect was eliminated when the culture media contained sulfhydryl donating reagents—glutathione and cysteine. This may offer a clue to a mechanism of ozone toxicity.