Earthworms play key roles in soils and sub-lethal effects of environmental toxicants on these organisms should be taken seriously, since they might have detrimental effects on higher ecological levels. In laboratory experiments we have assessed sub-lethal effects (body mass change and cast production) of imidacloprid on two earthworm species commonly found in different agricultural soils (Lumbricus terrestris and Aporrectodea caliginosa). After 7 days of exposure in contaminated soil, a significant loss of body mass was found in both species exposed to imidacloprid concentrations as low as 0.66 mg kg(-1) dry soil. These losses ranged from 18.3 to 39% for A. caliginosa and from 7.4 to 32.4% for L. terrestris, respectively. Changes in cast production, a new biomarker previously validated using L. terrestris, was assessed by soil sieving using the recommended mesh size (5.6 mm) for L. terrestris and three different mesh sizes for A. caliginosa (5.6, 4 and 3.15 mm). The 4 mm mesh size proved to be the most suitable sieve size for A. caliginosa. Cast production increased by 26.2% in A. caliginosa and by 28.1% in L. terrestris at the lowest imidacloprid concentration tested (0.2 mg kg(-1) dry soil), but significantly decreased at higher concentrations (equal to and above 0.66 mg kg(-1) dry soil) in both earthworm species after the 7 days exposure experiment. These decreases in cast production ranged from 44.5 to 96.9% in A. caliginosa and from 42.4 to 95.7% in L. terrestris. The EC(50) for cast production were 0.84 (L. terrestris) and 0.76 mg kg(-1) dry soil (A. caliginosa), respectively. The detected sub-lethal effects were found close to the predicted environmental concentration (PEC) of imidacloprid, which is in the range of 0.33-0.66 mg kg(-1) dry soil. The biomarkers used in the present study, body mass change and changes in cast production, may be of ecological relevance and have shown high sensitivity for imidacloprid exposure of earthworms. The measurement of changes in cast production should be considered for inclusion in current standard tests.