The role of ambient (21%) and physiological oxygen (2-5%) in the immortalization of fetal vs. adult human lung fibroblasts was examined. Growth in low oxygen and antioxidants extended the lifespan of both fetal and adult strains. As the ectopic expression of telomerase could immortalize adult lung fibroblasts cultured in ambient oxygen, the lifespan-shortening effects of 21% oxygen must have been largely limited to telomeres. By contrast, fetal lung fibroblasts could not be immortalized in ambient oxygen in spite of telomere elongation by telomerase, suggesting more widespread oxidative damage. The long-term culture requirements for the immortalization of WI-38 fetal lung fibroblasts included supplementation with N-(tert) butyl hydroxylamine, dexamethasone, zinc and vitamin B12, in addition to growth in physiological oxygen. The mechanisms regulating telomere shortening remain controversial. The present results suggest that both end-replication and oxidative damage events contribute to telomere shortening in lung fibroblasts in vitro. These observations emphasize the need for better analytical techniques to distinguish whether the correlation of short telomeres with disease and mortality in humans reflects the consequences of increased proliferation, telomere shortening as a result of oxidative damage or some combination of these processes.