To investigate the role of particle size on the oxidation, bioavailability, and adverse effects of manufactured Cu nanoparticles (NPs) in soils, we exposed the earthworm Eisenia ferida to a series of concentrations of commercially produced NPs labeled as 20- to 40-nm or < 100-nm Cu in artificial soil media. Effects on growth, mortality, reproduction, and expression of a variety of genes associated with metal homeostasis, general stress, and oxidative stress were measured. We also used X-ray absorption spectroscopy and scanning X-ray fluorescence microscopy to characterize changes in chemical speciation and spatial distribution of the NPs in soil media and earthworm tissues. Exposure concentrations of Cu NPs up to 65 mg kg(-1) caused no adverse effects on ecologically relevant endpoints. Increases in metallothionein expression occurred at concentrations exceeding 20 mg kg(-1) of Cu NPs and concentrations exceeding 10 mg kg(-1) of CuSO4. Based on the relationship of Cu tissue concentration to metallothionein expression level and the spatial distribution and chemical speciation of Cu in the tissues, we conclude that Cu ions and oxidized Cu NPs were taken up by the earthworms. This study suggests that oxidized Cu NPs may enter food chains from soil but that adverse effects in earthworms are likely to occur only at relatively high concentrations (> 65 mg Cu kg(-1) soil).