The effects of oxygen tension and reducing agents upon the sensitivity of Giardia lamblia to metronidazole in vitro were determined using two different assays. First, the parasites were exposed to drug under high or low oxygen tension in liquid medium with or without fresh cysteine and ascorbic acid. Survival was determined by colony assay. Second, the effect of thiol reducing agents was measured directly by colony assay in semi-solid media (low pO2 conditions). Trophozoites were strikingly (greater than 100-fold) less sensitive to metronidazole under the aerobic condition of the first assay. In contrast, the reducing agents had much lesser effects (2- to 5-fold) depending upon the assay. Finally, G. lamblia isolated from a patient treated with metronidazole unsuccessfully four times showed similar metronidazole sensitivity to a standard strain under both aerobic and reduced O2 tension conditions.