Coelomocytes comprise the immune system of earthworms and due to their sensitivity responding to a wide range of pollutants have been widely used as target cells in soil ecotoxicology. Recently, in vitro assays with primary cultures of coelomocytes based in the neutral red uptake (NRU) assay have been developed as promising tools for toxicity assessment chemical in a reproducible and cost-effective manner. However, NRU showed a bimodal dose–response curve previously described after in vivo and in vitro exposure of earthworm coelomocytes to pollutants. This response could be related with alterations in the relative proportion of coelomocyte subpopulations, amoebocytes and eleocytes. Thus, the aims of the present work were, first, to establish the toxicity thresholds that could be governed by different cell-specific sensitivities of coelomocytes subpopulations against a series of metals (Cu, Cd, Pb, Ni), and second to understand the implication that coelomocyte population dynamics (eleocytes vs. amoebocytes) after exposure to pollutants can have on the viability of coelomocytes (measured by NRU assay) as biomarker of general stress in soil health assessment. Complementarily flow cytometric analyses were applied to obtain correlative information about single cells (amoebocytes and eleocytes) in terms of size and complexity, changes in their relative proportion and mortality rates. The results indicated a clear difference in sensitivity of eleocytes and amoebocytes against metal exposure, being eleocytes more sensitive. The bimodal dose–response curve of NRU after in vitro exposure of primary cultures of coelomocytes to metals revealed an initial mortality of eleocytes (decreased NRU), followed by an increased complexity of amoebocytes (enhanced phagocytosis) and massive mortality of eleocytes (increased NRU), to give raise to a massive mortality of amoebocytes (decrease NRU). A synergistic effect on NRU was exerted by the exposure to high Cu concentrations and acidic pH (elicited by the metal itself), whereas the effects on NRU produced after exposure to Cd, Ni and Pb were due solely to the presence of metals, being the acidification of culture medium meaningless.