S-Carboxymethyl-L-cysteine (CMC) is used both as an orally administered mucolytic agent and as a probe drug for uncovering polymorphic sulfoxidation of other sulfur-containing drugs in humans. However, several recent studies could not confirm the formation of significant amounts of urinary sulfoxides of CMC or its decarboxylation product S-methyl-L-cysteine. The metabolism of CMC and a 13C-labeled isotopomer was therefore reinvestigated in 11 and 14 humans, respectively, and emphasis was laid on monitoring of potential alternative metabolic pathways. Combined capillary gas chromatography/electron impact or negative-ion chemical ionization mass spectrometry employing stable isotope-labeled analogues as internal standards were used for identification and quantification of CMC metabolites in human urine. Three nitrogen-free metabolites that were identified as thiodiglycolic acid (TDGA, mean: 19.8% of the dose/24 hr), thiodiglycolic acid sulfoxide (TDGA-SO, mean: 13.3% of the dose/24 hr), and (3-carboxymethylthio)lactic acid (TLA, mean: 2.1% of the dose/8 hr), cumulatively account for about one-third of the dose during a urinary collection period of 24 hr. In addition, trace amounts of both TDGA and TLA exist as endogenous components in urine from persons not administered exogenous CMC at levels of about 5 and 1 nmol/ml, respectively. Both major metabolites TDGA and TDGA-SO, that were not considered in previous sulfoxidation phenotyping, are predominantly excreted after 8 hr. These results demonstrate the existence of a pyruvate-like metabolic pathway and suggest the necessity of a revision of the hitherto accepted biotransformation route of CMC in humans.