Abstract Metals are detoxified and sequestered into subcellular compartments when accumulated by earthworms. Differential centrifugation was used to quantify subcellular Pb in three separate studies to measure 14-day acute toxicity (lethality), 28/56-day reproductive effects, and 90-day bioaccumulation in spiked-soil exposed earthworms, Eisenia fetida. Observed toxicity and total body Pb was consistent with published work of others. Pb showed concentration-dependent toxicity relationships (lethality and reproduction) for total and subcellular Pb. Toxic fraction and total Pb showed similar concentration–response patterns in the 14-day and 28/56-day studies and tended to increase towards a plateau at higher concentrations. Linear correlations of subcellular to total Pb was observed in all studies except the 90-day bioaccumulation study in which toxic fraction Pb appeared to approach a maximum over the period between Day 56 and Day 90. In a follow-on study using two different contaminated soil types, toxic fraction and total Pb concentrations as related to reproductive effects were consistent with data from our spiked soil studies, and this suggests it may be possible to use such values to “factor out” matrix-specific influences that otherwise skew toxicity values when expressed relative to soil concentrations. Our findings, however, suggest the subcellular fractionation approach may not offer advantages over total Pb determination in short-term exposure studies but may become important when longer exposure periods (greater than 90 days) are considered. In this respect, the technique we describe has the potential to provide valuable information for assessing and interpreting Pb toxicity as a function of earthworm body burden.