Low plant-availability of iron (Fe), zinc (Zn) and manganese (Mn) leads to micronutrient deficiency, causing significant yield reductions of crops throughout the world, especially in calcareous soils. This study was performed in order to evaluate the efficiency of xylem sap analysis in the determination of Fe, Zn and Mn availability in plants (Cucumis sativus L.) affected by calcium carbonate (CaCO3) levels. A soil with six levels of CaCO 3 (0-10% DW) was used. We performed a combination approach, including analysis of the soil mobility of micronutrients using different extractants (water, DTPA-TEA and ammonium acetate), as well as xylem and shoot elemental analysis. Generally, application of CaCO3 resulted in a pH increase of the bulk soil of 1.4-2.2 pH units; extractability of all micronutrients was significantly decreased 1.4-4.2 times, irrespective of the extracting solution. Xylem sap Fe, Zn and Mn concentrations were significantly correlated with the respective concentrations in the soil extracting solutions. By contrast, only shoot concentrations of Zn and Mn, but not of Fe, were linearly correlated with their extractable forms. With electrothermal atomic absorption spectrometry, changes in xylem sap concentrations of micronutrients were detected without preliminary mineralization of plant material, in contrast to shoot analysis. Our results demonstrate that xylem sap analysis offers the advantages of a simple characterization of multi-microelement availability in plants under CaCO3 stress.