The earthworm species Eisenia fetida and Eisenia andrei are commonly used in ecotoxicological standard tests. In the present study, we compared the sensitivity of E. fetida with that of two soil-dwelling earthworm species (Aporrectodea caliginosa and Lumbricus terrestris) in order to evaluate the capacity of E. fetida to predict effects of the insecticide imidacloprid. Responses were compared using two endpoints, a biochemical (changes in heat shock protein level (hsp70)) and a behavioural (avoidance behaviour). For the hsp70 analysis, the earthworms were exposed for 1, 7 and 14 days and the avoidance tests were conducted using a 48-h incubation time. The tested imidacloprid concentrations ranged from 0.2 to 4 mg kg(-1) soil dry weight (DW). E. fetida showed significant avoidance behaviour towards all test concentrations (0.2, 0.66 and 2 mg kg(-1) DW), while L. terrestris and A. caliginosa did not avoid imidacloprid-contaminated soil. Significant changes in hsp70 level in E. fetida occurred at the lowest concentration (0.2 mg kg(-1) DW and 14 days), while no effects were observed until exposure to 2 (A. caliginosa; after 1, 7 and 14 days) and 4 mg kg(-1) DW (L. terrestris; after 14 days). The present study revealed species-specific differences in sensitivity with E. fetida being the most sensitive species and L. terrestris the least sensitive. Moreover, some of the observed effects were detected at environmentally relevant concentrations. Our study indicated different sensitivities between closely related species highlighting the importance of using a multiple selection of species in ecotoxicology to predict harmful environmental effects more accurately and minimise underestimations. In the case of testing only one species or one trophic level, a further increase of safety factors is advisable. Moreover, it can be concluded that hsp70 was not a good indicator of imidacloprid toxicity given the low induction for the selected test species. The results of the present study highlight the species dependency of the avoidance test in case of imidacloprid.