Mercury (Hg) fractionation, speciation, bioavailability, and ecotoxicity were investigated in three highly contaminated soils from chlor-alkali plants. Single extractions and a validated four-step sequential extraction scheme were used. Total, volatile, and methyl-Hg concentrations were determined. Mercury was then separated in fractions defined as water-soluble (F1), exchangeable (F2), organic (F3), and residual (F4). Germination and growth inhibition of barley (Hordeum vulgare) and mortality of earthworms (Eisenia andrei) were assessed, and tissue-Hg concentrations of exposed organisms were determined. Results revealed highly (295 +/- 18-11,500 +/- 500 microg Hg/g) contaminated soils, but extracted fractions indicated relatively low mobility of Hg. Nevertheless, the water-soluble and the CaCl2-extractable fractions represented significant Hg concentrations (299 +/- 18 microg/g in soil 3, 67.4 +/- 2.3 microg/g in soil 1, and 9.5 +/- 0.3 microg/g in soil 2), and volatile Hg ranged between 14 and 98% of total Hg. Overall, Hg concentrations reached 6,560 +/- 240 microg/g in roots, 4,200 +/- 1,070 microg/g in aerial plants, and 1,410 +/- 120 microg/g in E. andrei. Earthworm mortality was 100% after exposure to the soil with the highest concentration of mobile Hg. In the latter soil, earthworm fragmentation and chlorotic plants were observed. Bioconcentration factors (BCFs) were higher in barley compared to earthworms, but BCFs yielded misleading values after exposure to the extremely contaminated soil. This study shows that Hg accumulated primarily in the roots, but results also indicate uptake of gaseous Hg by the aerial plants of barley. Tissue-Hg concentrations of both exposed organisms were correlated with water-soluble and CaCl2-extractable Hg, and growth inhibition was in agreement with Hg fractionation.