Hypoxia and amino acid deprivation downregulate expression of extracellular matrix genes in lung fibroblasts. We examined the effect of hypoxia on amino acid uptake and protein formation in human lung fibroblasts. Low O₂ tension (0% O₂) suppressed incorporation of [³H]proline into type I collagen without affecting [³⁵S]methionine labeling of other proteins. Initial decreases in intracellular [³H]proline incorporation occurred after 2 h of exposure to 0% O₂, with maximal suppression of intracellular [³H]proline levels at 6 h of treatment. Hypoxia significantly inhibited the uptake of radiolabeled proline, 2-aminoisobutyric acid (AIB), and 2-(methylamino)isobutyric acid (methyl-AIB) while inducing minor decreases in leucine transport. Neither cycloheximide nor indomethacin abrogated hypoxia-related suppression of methyl-AIB uptake. Efflux studies demonstrated that hypoxia inhibited methyl-AIB transport in a bidirectional fashion. The downregulation of amino acid transport was not due to a toxic effect; function recovered on return to standard O₂ conditions. Kinetic analysis of AIB transport revealed a 10-fold increase in K_m accompanied by a small increase in maximal transport velocity among cells exposed to 0% O₂. These data indicate that low O₂ tension regulates the system A transporter by decreasing transporter substrate affinity.