High concentrations of O₂ inhibit epithelial cell proliferation that resumes on recovery in room air. To determine whether growth arrest is mediated by transforming growth factor- (TGF-), changes in cell proliferation during exposure to hyperoxia were assessed in the mink lung epithelial cell line Mv1Lu and the clonal variant R1B, which is deficient for the type I TGF- receptor. Mv1Lu cells treated with TGF- accumulated in the G₁ phase of the cell cycle as determined by propidium iodide staining, whereas proliferation of R1B cells was unaffected by TGF-. In contrast, hyperoxia inhibited proliferation of both cell lines within 24 h of exposure through an accumulation in the S phase. Mv1Lu cells treated with TGF- and exposed to hyperoxia accumulated in the G₁ phase, suggesting that TGF- can inhibit the S phase accumulation observed with hyperoxia alone. Cyclin A was detected in cultures exposed to room air or growth arrested by hyperoxia while decreasing in cells growth arrested in the G₁ phase by TGF-. Finally, hyperoxia failed to activate a TGF--dependent transcriptional reporter in both Mv1Lu and R1B cells. These findings reveal that simple growth arrest by hyperoxia involves a defect in S phase progression that is independent of TGF- signaling.