© 2016 Borrego et al. Background: The majority of cancer cells have a unique metabolic requirement for methionine that is not observed in normal, non-tumorigenic cells. This phenotype is described as "methionine dependence" or "methionine stress sensitivity" in which cancer cells are unable to proliferate when methionine has been replaced with its metabolic precursor, homocysteine, in cell culture growth media. We focus on the metabolic response to methionine stress in the triple negative breast cancer cell line MDA-MB-468 and its methionine insensitive derivative cell line MDA-MB-468res-R8. Results: Using a variety of techniques including fluorescence lifetime imaging microscopy (FLIM) and extracellular flux assays, we identified a metabolic down-regulation of oxidative phosphorylation in both MDA-MB-468 and MDA-MB- 468res-R8 cell types when cultured in homocysteine media. Untargeted metabolomics was performed by way of gas chromatography/time-of-flight mass spectrometry on both cell types cultured in homocysteine media over a period of 2 to 24 h. We determined unique metabolic responses between the two cell lines in specific pathways including methionine salvage, purine/pyrimidine synthesis, and the tricarboxylic acid cycle. Stable isotope tracer studies using deuterium-labeled homocysteine indicated a redirection of homocysteine metabolism toward the transsulfuration pathway and glutathione synthesis. This data corroborates with increased glutathione levels concomitant with increased levels of oxidized glutathione. Redirection of homocysteine flux resulted in reduced generation of methionine from homocysteine particularly in MDA-MB-468 cells. Consequently, synthesis of the important one-carbon donor S-adenosylmethionine (SAM) was decreased, perturbing the SAM to S-adenosylhomocysteine ratio in MDA-MB-468 cells, which is an indicator of the cellular methylation potential. Conclusion: This study indicates a differential metabolic response between the methionine sensitive MDA-MB-468 cells and the methionine insensitive derivative cell line MDA-MB-468res-R8. Both cell lines appear to experience oxidative stress when methionine was replaced with its metabolic precursor homocysteine, forcing cells to redirect homocysteine metabolism toward the transsulfuration pathway to increase glutathione synthesis. The methionine stress resistant MDAMB- 468res-R8 cells responded to this cellular stress earlier than the methionine stress sensitive MDA-MB468 cells and coped better with metabolic demands. Additionally, it is evident that S-adenosylmethionine metabolism is dependent on methionine availability in cancer cells, which cannot be sufficiently supplied by homocysteine metabolism under these conditions.