The metabolism of the organophosphonate herbicide glyphosate was investigated in 163 environmental bacterial strains, obtained by a variety of isolation strategies from sites with or without prior exposure to the compound. Isolates able to use glyphosate as sole phosphorus source were more common at a treated site, but much less abundant than those capable of using the glyphosate metabolite aminomethylphosphonic acid (AMPA). Nevertheless, all 26 strains found to metabolise the herbicide did so via an initial cleavage of its carbon-phosphorus bond to yield sarcosine; no evidence for its metabolism or co-metabolism to AMPA was obtained.