We investigated the nitrous oxide-induced inactivation of methionine synthase and the concurrent homocysteine (Hey) export in mutant fibroblasts with defects in the homocysteine catabolizing enzyme, cystathionine β-synthase, or in methionine synthase, which carries out homocysteine remethylation. The fibroblasts were incubated in various concentrations of methionine to create conditions favoring methionine conservation or catabolism. In cystathionine β-synthase-deficient cells, high medium methionine partly protected the enzyme against inactivation, as previously found in normal fibroblasts. The Hey export rate at low methionine levels was low (0.2–0.6 nmol/ h/106 cells), and increased 2–3-fold at high methionine levels. Nitrous oxide enhanced Hey export rate at low methionine, so that in the presence of nitrous oxide, the Hey export became less dependent of methionine. In cblG cells, the enzyme inactivation was moderate and independent of medium methionine. The Hey export rate was intermediate (0.5–0.8 nmol/h/106 cells) at low methionine levels, and increased moderately (<2-fold) at high methionine levels or following nitrous oxide exposure. In cblE mutants, the enzyme activity was not affected by nitrous oxide, and the Hey export was high (0.8–1.6 nmol/h/106 cells) and independent of methionine and nitrous oxide. These data suggest that Hey remethylation and cystathionine βsynthase activity are major determinants of Hey export at low and high methionine, respectively. The low susceptibility of methionine synthase to nitrous oxide in the presence of high methionine or in cblG or cblE mutants is probably related to low catalytic turnover.