Imidacloprid (1-[(6-chloro-3-pyridinyl)methyl]-N-nitro-2-imidazolidinimine), a chloronicotinyl insecticide used to control biting and sucking insects, is very persistent in the soil with a half-life often greater than 100 days. Our objectives were to 1) discover, isolate, and characterize a single microbial species with the ability to degrade imidacloprid and 2) determine the persistence of imidacloprid in Drummer and Exeter soils in order to determine the bioavailability of imidacloprid. Soil-free stable enrichments in N-limited media were obtained which degraded 19 mg L−1 (43%) and 11 mg L−1 (16%) of the applied imidacloprid and produced about 19 mg L−1 6- chloronicotinic acid in three weeks. Cell free controls had no loss of imidacloprid. Strain PC-21, obtained from the enrichment cultures degraded 37% to 58% of 25 mg L−1 imidacloprid in tryptic soy broth after three weeks. Trace amounts of N03-/NO2- were produced and six metabolites were characterized by HPLC and LC-MS. 6-chloronicotinic acid was not produced. Less than 1% of the applied 14C was incorporated into the microbial biomass. No 14CO2 was detected. Strain PC-21 cometabolizes imidacloprid. PCR amplification of a 500 bp sequence of 16s rRNA identified strain PC-21 to be of the genus Leifsonia. Soils were treated in triplicate with either 0.1 mg kg−1 14C-methylene-imidacloprid or 1.0 mg kg−1 non-radiolabeled imidacloprid. Cumulative 14CO2 was less than 1.5% of applied 14C in all soils over 400 days. Soils treated with 14C-benzoic acid mineralized greater than 40% of this compound after 21 days. Drummer surface soil was able to mineralize greater than 60% of 14C-2, 4-D after 40 days, while the other soils mineralized less then 5%. To determine imidacloprid bioavailability, sequential extractions were carried out. Water-, acetonitrile-, and acid-extractable fractions were collected. Less than 10% of the imidacloprid was degraded in the Drummer subsurface soil compared to about 40% in the Drummer surface soil and about 50% in both Exeter soils. Between 70% and 99% of imidacloprid in Exeter surface soil was present in the water-extractable fraction, compared to only 12% in the Drummer. Subsurface soils produced similar results, with 25% of the imidacloprid remaining after 400 days in a water-extractable form in the Drummer soil.