Trifluralin (alpha,alpha,alpha-trifluoro-2,6-dinitro-N,N-dipropyl-p-toluidine; TFL) is a pre-emergence, soil-incorporated herbicide that has been in agricultural use since the early 1960s and is moderately persistent in soil. The purpose of this study was to isolate and characterise TFL-resistant bacteria from a soil in which this pesticide has been used for the last four decades and to determine their ability to degrade TFL using HPLC. Eight bacteria were isolated by repeated subculture in liquid medium with TFL as carbon source and a ninth (isolate 9) from growth around TFL crystals on solid medium. The bacteria from enriched liquid culture were identified by biochemical tests and 16S rDNA sequencing. In a mineral salts medium with 0.1% succinate, 0.1% yeast extract and 50 mg l-1 TFL, reductions in the level of pesticide of 24.6% for Klebsiella sp., 16.4% for Herbaspirillum sp., 25.0% and 16.0% for two strains of Bacillus sp. and 21.0% for unidentified isolate number 9 were obtained after 30 days. These were similar to the level obtained using a known TFL-degrading bacterium, Brevundimonas diminuta (NCIMB 10329). Three Pseudomonas sp. and one Bacillus sp. reduced levels by less than 5%. The five positive isolates can be used to study the biochemical and molecular biology of TFL biodegradation with the aim of optimising the degradative ability of one or more of the isolates for future use in bioremediation processes. (c) Biosciences Information Services.