The metabolism of glyphosate [N-(phosphonomethyl)glycine] in a bacterium tentatively identified as an Arthrobacter sp., capable of growth on this herbicide as its sole phosphorus source, has been investigated using solid-state NMR techniques as well as radiotracer analysis. The pathway involves the conversion of glyphosate to glycine, a C1 unit and phosphate. The phosphonomethyl carbon is specifically incorporated into the amino acids serine, cysteine, methionine, and histidine, as well as into purine bases and thymine, indicating the involvement of tetrahydrofolate in single-carbon transfer reactions. Glycine derived from glyphosate is utilized in purine and protein biosynthesis. This pathway for glyphosate degradation in a gram-positive bacterium is similar to that previously reported for Pseudomonas sp. PG2982 [Jacob et al. (1985) J. Biol. Chem. 260, 5899-5905] and is distinct from that reported for soil metabolism of glyphosate where aminomethylphosphonic acid has been shown to be a major metabolite. Preliminary evidence is presented which indicates that the conversion of glyphosate to glycine and the C1 unit involves the intermediate formation of sarcosine. Thus, the primary event in glyphosate degradation by Arthrobacter sp. GLP-1 is the cleavage of its C-P bound. This report constitutes the first demonstration of the metabolism of glyphosate in a gram-positive bacterium.