S-Adenosylmethionine (SAM, AdoMet) is the most important methyl donor used for synthesis of nucleic acids, phospholipids, creatine, and polyamines and for methylation of many bioactive molecules. The metabolic response of the lung to oxidative stress of hyperoxia requires increased RNA and protein synthesis for energy metabolism, growth arrest, and antioxidant defense. We studied the production of SAM and other aspects of methionine metabolism in lung epithelial cells exposed to hyperoxia. Human lung epithelial-like (A549) and primary small airway epithelial (SAE) cells were exposed to normoxia (21% O2) or hyperoxia (95% O2). Cell methionine and S-adenosylmethionine content increased in response to hyperoxia in SAE and A549 cells. Because methionine adenosyl transferase (MAT) is the rate-limiting enzyme of the pathway, we examined the expression of a lung epithelial isoform of MAT 2A in hyperoxia. Western blots revealed a novel MAT 2A isoform expressed in both cell types, with a lower molecular mass than that described in Jurkat cells. Cloning and sequencing of the MAT 2A cDNA revealed one silent nucleotide substitution compared to that expressed in Jurkat. The lower mass of MAT 2A in both lung epithelial cells indicated that the absence of the major posttranslational modification of MAT 2A found in Jurkat. MAT 2A protein progressively increased during hyperoxic exposure in both transformed and primary lung epithelium. Increased flux of 13C-labeled methionine to S-adenosylhomocysteine (SAH) in A549 demonstrated that SAM's methyl group was utilized, and increased formation of cystathionine indicated that at least part of SAM generated was directed toward cysteine/GSH in the transsulfuration pathway. These results indicate activation of MAT 2A and the transmethylation pathway in the metabolic response to hyperoxia in lung epithelium. © 2005 Elsevier Inc. All rights reserved.