Monitoring strategies were developed re track non-genetically engineered Pseudomonas putida strains in the open environment. The strain El was used for four years for the biodegradation of phenolic compounds in industrial wastewater in Polva, Estonia. In this study we used the strain E2 which is a non-carbenicillin-resistant variant of the strain E1. Both strains have a deletion of approximately 34 kb in the TOL plasmid pWWO which served as a basis for discrimination from indigenous bacteria by molecular analyses. Other targets used for PCR and DNA hybridization were the xylE gent and a sequence located in the left-handed region of to the transposon Tn4652. In laboratory tests we demonstrated that two cells inoculated into 20 ml of river water could be detected against a background of mere than 10(7) colony forming units (CFUs) by a combination of growth on selective media and molecular analysis. Using the same combination of methods in a deliberate release experiment, detection of the released strain was possible only to 32 h after release. It is assumed that the released strains did not survive in the aquatic ecosystem, mainly due to the high dilution rate. The combination of cultivation on selective media and molecular analyses proved useful for tracking Pseudomonas putida strain E2 in an aquatic environment.