Evolution of resistance to heavy metals has been reported for several populations of soil living organisms occurring at metal contaminated sites. Such genetically based and heritable resistance contribute to the persistence of populations in contaminated areas. Here we report on molecular responses to experimental copper in populations of the earthworm, Dendrobaena octaedra, originating from copper contaminated soil near Gusum (Sweden) where heavy metal pollution has been present for several decades. We studied gene expression of six genes potentially involved in resistance to copper toxicity using F2-generations of D. octaedra populations, originating from reference sites and contaminated (High, Medium and Low) sites around Gusum. The main result was different expression patterns of genes encoding for two different isoforms (mt1 and mt2) of metallothionein proteins during experimental exposure to copper contaminated soil. Expression of mt1 showed a fast and significant upregulation in the High population and a slower, albeit significant, upregulation in Medium and Low populations. However, in the three reference populations no upregulation were seen. In comparison, a fast upregulation was also seen for the High population in the isoform mt2, whereas, gene expression of all other populations, including reference populations, showed slower upregulation in response to experimental copper. The results indicate that copper resistance in D. octaedra from contaminated areas is related to an increased expression of metallothioneins.