Understanding the relationships between accumulated metal speciation in cells and tissues of ecologically significant taxa such as earthworms will improve risk assessments. Synchrotron-based μ-focus X-ray spectroscopy was used to detect, localize, and determine ligand-speciation of Zn and Pb in thin sections of two epigeic earthworm species collected from a Pb/Zn-mine soil. The findings indicated that Zn and Pb partition predominantly as typical hard acids (i.e., strong affinities for O-donors) within liverlike chloragocytes. Moreover, Zn speciation was very similar in the chloragog and intestinal epithelia but differed subtly in the kidneylike nephridial tubules; neither Zn nor Pb was detectable in the ventral nerve cord. High resolution X-ray mapping of high pressure-frozen, ultrathin, freeze-substituted sections in a transmission electron microscope (TEM), combined with conventional TEM structural analysis, identified a new cell type packed with highly organized rough endoplasmic reticulum and containing deposits of Cd (codistributed with S); there was no evidence that these cells are major depositories of Zn or Pb. These data may be used in a systems biology approach to assist in the interpretation of metal-evoked perturbations in whole-worm transcriptome and metabolome profiles.