Exposure to cadmium poses a considerable risk to human health and environmental safety. Earthworms reside in the most contaminated sites on earth, displaying a phenomenal tolerance to toxic heavy metals. They exhibit a distinct metabolic pathway that allows the bio-accumulation of cadmium to yield body burdens in excess of 1/1000th of total dry body weight, a most impressive figure by any standard. However, the precise molecular mechanism underlying this phenomenon remains to be unraveled. This study meets this challenge by fully characterizing the major metal-binding protein in earthworms, namely the two isoforms of metallothionein. Chemical analysis of recombinant protein showed that although both isoforms bind equimolar amounts of cadmium (6 mol), wMT-2 is more stable during proton competition. Furthermore, isoform-specific transcript analysis demonstrated that only wMT-2 is responsive to cadmium in a dose and temporal manner. The specific sequestration of cadmium to wMT-2 protein was confirmed in situ using polyclonal antisera. The latter also provided the means for mapping the cellular and intracellular distribution of metallothionein, thus yielding a holistic insight into its involvement in cadmium transit during absorption, storage, and excretion. The structure-function relationship of wMT-2 and its role in cadmium detoxification through sequestration and compartmentalization is discussed.Earthworms are central to soil quality and fertility. The first authoritative account of the central role of the earthworm in soil formation, quality, and fertility dates back to Charles Darwin well over 100 years ago (1). More recent studies have shown this oligochaete annelid exhibiting an exceptional tolerance to cadmium, residing in soils with concentrations exceeding 600 g of cadmium/g dry weight (2). In addition to surviving this toxicological challenge, they bio-accumulate this metal ion to a body burden in excess of 1 mg/g dry weight (2, 3). This accumulation of cadmium is in stark contrast to the exclusion observed with other metal ions, for example, copper (4). However, even where ions with closely related chemistry, such as cadmium and zinc, are elevated within the same environment, the compartmentalization, and therefore the metabolic pathway, remains distinct (5). Therefore, the earthworm must have a highly developed and specific trafficking pathway for this toxic metal ion. Ultrastructural mapping has shown that the majority of the cadmium is sequestered as a thiol-based bioinorganic complex within intracellular vesicles in the earthworm's primitive liver-like tissue, the chloragog (5).The co-localization of cadmium and sulfur within a distinct intracellular organelle suggested the involvement of a sulfurrich protein. Metallothioneins (MTs) 1 are cysteine-rich cationic proteins that have been linked to cadmium detoxification in a wide range of phylogenetic orders (e.g. humans (6), fish (7), Caenorhabditis elegans (8), springtails (9), and others). However, until recently (2) no molecular information has b...