Two isolates of Pseudomonas sp., GLC11 and PG2982, can use glyphosate as a sole source of phosphorus. This ability is indicative of enzymatic cleavage of a carbon-phosphorus bond, and the enzyme has been named C-P lyase. We have cloned, in Escherichia coli, gene/s coding for C-P lyase on a broad host range cosmid pLA2917. Restriction fragment arrangement of cloned fragments of PG2982 and GLC11 has been established. Analysis by Southern hybridization between two clones revealed a strong homology between three PstI fragments of pPG-CP-14 (derived from PG2982) and pGC-CP-4 (derived from GLC11). With the construct pGC-CP-4 as a probe, the presence of a cryptic allele for C-P lyase has been demonstrated on the chromosome of the parent isolate, Pseudomonas aeruginosa PAO1. It is suggested that genetic rearrangement such as frame shift or a point mutation activated the cryptic C-P lyase gene. Metabolism of glyphosate by E. coli carrying pPG-CP-14 or pGC-CP-4 has been demonstrated by radiometric experiments.