The fate and impact of heavy metals produced by agricultural or industrial activities has become a crucial environmental problem. Bio-remediation processes of contaminated soils are in progress but there is still a strong need for tools assessing the evolution of the risk associated with the remaining pollutant concentrations i.e. the toxicity parameter. Among these tools, biosensors are of high interest as they give real heavy metal bio-availability information, i.e. the concentrations really perceived by organisms in soil. Such tools can provide a better understanding of the factors controlling toxicity mechanisms in natural soil and thus improve bio-remediation approaches. The aim of this work was to apply a microcosm approach to evaluate the effect of OM inputs on copper toxicity and mobility. A Copper Specific Electrode (CSE) was applied to measure the concentration of free and complexed Cu. A whole-cell biosensor was used to assess copper bio-availability in a vineyard Soil amended with different types of Organic Matter (SOM). The whole-cell copper biosensor is a Pseudomonas fluorescens strain that was kindly provided by O. Nilbroe (2001). The P. fluorescens strain (DF57-Cu15) was selected as a Cu reporter due to its high specific response and tolerance to Cu in pure culture. This DF57-Cu15 copper sensor responds to Cu-polluted soil leachates in a concentration-dependent manner. Regulation of the expression of the reporter proteins is related to the amount of copper ions available to the bacteria. CSE and biosensor Cu measurements were applied to Ca(NO3)2 leachates of two vineyard soils (a sand and a silty clay loam) to compare the free and bio-available copper concentrations as affected by organic matter inputs. In the sandy soil, the OM content presented only a slight effect on Cu speciation, soil pH being the major controlling factor. In the silty clay loam a good relation was observed between soil organic matter content and Cu speciation. Higher exchangeable, free and bio-available copper concentrations were observed in the leachates of the control soil not amended with organic matter demonstrating the sink effect of applied OM. The higher Cu bio-availability in the control soils was strongly correlated to the higher Cu toxicity revealed by the increase of copper resistant bacteria. In more detail, bio-available and free Cu do not correlate linearly in the different soils. Thus, the bio-available copper concentrations were about 4 times lower than the total exchangeable copper for the two organic matter amended soils, whereas they presented similar bio-available and water-exchangeable copper concentrations in the non amended control soil. These results confirmed that bio-available heavy metals concentration can vary to a large extend in natural soils dependent from the amount and type of organic matter, but also pH. This study also demonstrates that the correlation between the bio-available copper and water-exchangeable and free copper concentrations, often used in risk assessment studies, is not straightforward. This confirms the necessity to use bio-reporters to provide more realistic information on the available, and thus toxic status of the metal in the soil. Significant improvements in risk assessment studies may be expected from the development and use of such sensors.. Nybroe et al. FEMS Microbiol. Ecol. 38 : 59-67.