Because boron (B) and calcium (Ca2+) seem to have a strong effect on legume nodulation and nitrogen fixation, rhizobial symbiosis with leguminous plants, grown under varying concentrations of both nutrients, was investigated. The study of early pre-infection events included the capacity of root exudates to induce nod genes, and the degree of adsorption of bacteria to the root surface. Both phenomena were inhibited by B deficiency, and increased by addition of Ca2+, resulting in an increase of the number of nodules. The infection and invasion steps were investigated by fluorescence microscopy in pea nodules harbouring a Rhizobium leguminosarum strain that constitutively expresses green fluorescent protein. High Ca2+ enhanced cell and tissue invasion by Rhizobium, which was highly inhibited after B deficiency. This was combined with an increased B concentration in nodules of plants grown on B-free medium and supplemented with high Ca2+ concentrations, and that can be attributed to an increased B import to the nodules. Histological examination of indeterminate (pea) and determinate (bean) nodules showed an altered nodule anatomy at low B content of the tissue. The moderate increase in nodular B due to additional Ca2+ was not sufficient to prevent the abnormal cell wall structure and the aberrant distribution of pectin polysaccharides in B-deficient treatments. Overall results indicate that the development of the symbiosis depends of the concentration of B and Ca2+, and that both nutrients are essential for nodule structure and function.