Metal ions in excess of metabolic requirements are potentially toxic and must be removed from the vicinity of important biological molecules to protect organisms from adverse effects. Correspondingly, metals are sequestrated in various forms, defining the accumulation pattern and the magnitude of steady-state levels reached. To investigate the subcellular fractions over which Ca, Mg, Fe, Cu, Zn, Cd, Pb, Ni, and As are distributed, earthworms (Aporrectodea caliginosa) collected from the field were analyzed by isolating metal-rich granules and tissue fragments from intracellular microsomal and cytosolic fractions (i.e., heat-stable proteins and heat-denatured proteins). The fractions showed metal-specific binding capacity. Cadmium was mainly retrieved from the protein fractions. Copper was equally distributed over the protein fraction and the fraction comprising tissue fragments, cell membranes, and intact cells. Zinc, Ca, Mg, and As were mainly found in this fraction as well. Lead, Fe, and Ni were mainly isolated from the granular fraction. To study accumulation kinetics in the different fractions, three experiments were conducted in which earthworms were exposed to metal-spiked soil and a soil contaminated by anthropogenic inputs and, indigenous earthworms were exposed to field soils. Although kinetics showed variation, linear uptake and steady-state types of accumulation patterns could be understood according to subcellular compartmentalization. For risk assessment purposes, subcellular distribution of metals might allow for a more precise estimate of effects than total body burden. Identification of subcellular partitioning appears useful in determining the biological significance of steady-state levels reached in animals. © 2006 SETAC.