There is in vitro evidence to support the notion that directed migration (chemotaxis) is involved in the recruitment of alveolar macrophages in vivo. Because O2 is widely used in the treatment of pulmonary diseases, we examined the effect of hyperoxia on migration of guinea pig alveolar macrophages in vitro. Migration was measured in blind-well chambers incubated in either room air or hyperoxia. N-formyl-methionyl-phenylalanine was used to stimulate random migration and to produce directed migration. Migration was quantified by counting the number of mononuclear cells per oil immersion field that had migrated completely through a polycarbonate filter with 5-micrometer pores. The average PO2 in the cell suspensions incubated in room air was 100 mm Hg. In the hyperoxic environments, the average PO2 at 1 h was 260 mm Hg, whereas at 2 and 3 h, it was 410 and 425 mm Hg, respectively. In 6 separate experiments, there was no significant difference between the mean response to N-formyl-methionyl phenylalanine in hyperoxia and in room air after 1 h of incubation. After 2 and 3h of incubation, however, the response in hyperoxia was significantly (P less than 0.002) lower than that in room air. The decreased response in hyperoxia did not appear to result from loss of viability of responding cells, diminished adherence of cells to the filters, loss of activity of N-formyl-methionyl phenylalanine exposed to high PO2, or failure of the cells to exhibit directed migration. Instead, it appeared that hyperoxia decreased the response of alveolar macrophages primarily by impairing random migration.