The increasing use of zinc oxide nanoparticles (ZnO NPs) in agriculture renders it necessary to evaluate their impact on soil non-target organisms. This work studies Zn availability to earthworms from the ZnO (NP and bulk) applied to two agricultural soils with a different pH at 20, 225, 500, and 1000 mg Zn kg -1 . Zn uptakes and the effects on Eisenia andrei, grown under controlled conditions, were determined. Effects were assessed at three levels: organisms, mortality, growth and reproduction; biochemical, catalase and glutathione S-transferase activities, malondialdehyde (MDA), and protein content; cellular in coelomocytes, reactive oxygen species (ROS) generation, lysosomal membrane alterations (RN) and mitochondrial dysfunction (MTT). Available Zn was 100-fold higher in acidic than in calcareous soil and did not differ among ZnO (NP or bulk). Zn in worms was auto-regulated regardless of the soil Zn concentration, pH and ZnO size. Effects on mortality and weight were observed only in the acidic soil at the highest concentration, ZnO NPs reduced survival and body weight, while ZnO bulk reduced body weight. Reproduction parameters in acidic soil were: EC50 (fecundity) 277 and 256 mg Zn kg-1 and EC50 (fertility) 177 and 179 mg Zn kg-1 for ZnO NPs and bulk, respectively, with no found NP-specific effects. No responses of enzymatic activities, MDA and MTT were detected. ROS and RN were altered in the coelomocyte cells of earthworms in the two soils, but effects depended on ZnO size suggesting nanospecific effects. Soil pH governs toxicity more than ZnO size regardless of body Zn concentration., (Copyright © 2019 Elsevier Ltd. All rights reserved.)