Thirty-four strains of Pseudomonas pseudomallei isolated from soil were selected for their ability to degrade the phosphonate herbicide glyphosate. All strains tested were able to grow on glyphosate as the only phosphorus source without the addition of aromatic amino acids. One of these strains, P. pseudomallei 22, showed 50% glyphosate degradation in 40 h in glyphosate medium. From a genomic library of this strain constructed in pUC19, we have isolated a plasmid carrying a 3.0-kb DNA fragment which confers to E. coli the ability to use glyphosate as a phosphorus source. This 3.0-kb DNA fragment from P. pseudomallei contained two open reading frames (glpA and glpB) which are involved in glyphosate tolerance and in the modification of glyphosate to a substrate of the Escherichia coli carbon-phosphorus lyase. glpA exhibited significant homology with the E. coli hygromycin phosphotransferase gene. It was also found that the hygromycin phosphotransferase genes from both P. pseudomallei and E. coli confer tolerance to glyphosate.