Kanamycin (Km)-resistant bacterial populations in different soil, river water, sewage and pig manure slurry samples were enumerated and their prevalence in the total populations determined. About 350 Km-resistant Gram-negative colonies grown in the presence of kanamycin were identified using a rapid presumptive identification scheme. They were then screened for the presence of Tn5 and nptII sequences using hybridization of cells in dot blots, of Southern-blotted genomic DNA extracts and of PCR amplification products. Colonies reacting positively with a 2.7 kb probe of the central region of Tn5, or with a 925 bp nptII specific probe were primarily obtained from sewage samples, whereas fewer were obtained from pig manure slurry, river water and soil. However, in soil samples bacteria containing Tn5 or nptII were not found. Transposon Tn5 carrying the nptII gene could be unequivocally demonstrated in 3 isolates from sewage, identified as Aeromonas spp. (2x) and Escherichia coli. HindIII digests of chromosomal DNA obtained from these strains were cloned and shown to confer Km resistance to a sensitive E. coli strain. Further, various strains revealed the presence of nptII homologous sequences in a non-Tn5 background. The occurence of Tn5 and nptII in the samples was also assessed via PCR analysis of total community DNA extracts obtained from the aforementioned environmental samples. Evidence for the occurence of nptII was obtained for sewage, pig manure slurry, for 2 (out of 3) river water (Avon, Rhine) and 3 (out of 6) soil (Flevo silt loam, Westmaas silt loam, Ahlum rhizosphere) samples. Tn5 was not detectable via PCR in any of these environmental DNA extracts but it was found in Ede loamy sand and Flevo silt loam samples taken from a field microplot 2 and 4 weeks after release of a Tn5-containing genetically modified organism.