The metabolism of glycine into glutathione was monitored noninvasively in vivo in intact rat mammary adenocarcinomas (R3230Ac) by MRI and MRS. Metabolism was tracked by following the isotope label from intravenously infused [2‐^13^C]‐glycine into the glycinyl residue of glutathione. Signals from [2‐^13^C]‐glycine and γ‐glutamylcysteinyl‐[2‐^13^C]‐glycine (^13^C‐glutathione) were detected by nonlocalized ^13^C spectroscopy, as these resonances are distinct from background signals. In addition, using spectroscopic imaging methods, heterogeneity in the in vivo tumor distribution of glutathione was observed. In vivo spectroscopy also detected isotope incorporation from [2‐^13^C]‐glycine into both the 2‐ and 3‐carbons of serine. Analyses of tumor tissue extracts showed single‐ and multiple‐label incorporation from [2‐^13^C]‐glycine into serine from metabolism through the serine hydroxymethyltransferase and glycine cleavage system pathways. Mass spectrometric analysis of extracts also showed that isotope‐labeled serine is further metabolized via the trans‐sulfuration pathway, as ^13^C isotope labels appear in both the glycinyl and cysteinyl residues of glutathione. Our studies demonstrate the use of MRI and MRS for the monitoring of tumor metabolic processes central to oxidative stress defense. Copyright © 2011 John Wiley & Sons, Ltd.