Several bacterial strains are known to metabolize s-triazines herbicides among which atrazine reported worldwidel to contaminate soil and water resources. The complete mineralization of atrazine to carbon dioxide and ammonium is catalyzed by 6 hydrolytic enzymes encoded by atzABCDEF genes located on the plasmid pADP1 in Pseudomonas ADP. The atzABC genes code for the upper pathway transforming atrazine to cyanuric acid, while the atzDEF genes code for the lower pathway transforming cyanuric acid to simple compounds. In order to study the genetic plasticity of the atrazine catabolic pathway, we performed an in vitro evolution study consisting in applying a continuous selection pressure with cyanuric acid as sole nitrogen source on Pseudomonas sp. After in vitro evolution the cyanuric acid degradation potential using HPLC analysis, of the native and of the newly-evolved Pseudomonas sp. population was assessed. Characterization of genetic potential revealed that the atzABC sequences were deleted in the newly-evolved population, probably by homologous recombination mediated by IS sequences. As a consequence, this in vitro evolution experiment suggests that the selection pressure exerted by the cyanuric acid led to the selection of a population showing a better fitness towards this metabolite, due to the loss of a genetic overload.