1α,25-dihydroxy vitamin D3 [1α,25(OH)2D3], the hormonal form of vitamin D3 that is essential for the maintenance of calcium-phosphorus homeostasis in birds and mammals, has been found in several plants. In order to study the metabolic role of 1α,25(OH)2D3 in plant cells, we monitored the changes of cellular levels of 1α,25(OH)2D3 in cell suspension cultures of Solanum malacoxylon Sendt. under different experimental conditions. We also measured the levels of cholesterol, the assumed precursor of vitamin D3, and other end-product sterols such as β-sitosterol, stigmasterol and campesterol. It was found that when cells are incubated in a calcium-deprived medium, the level of 1α,25(OH)2D3 increases markedly within a few hours. Treatment with methyl jasmonate (MeJ), a linolenic acid-derived compound that accumulates during wounding stress, was found to reduce the cellular level of 1α,25(OH)2D3 and to promote its secretion into the culture medium. After treatment with cellulysin, a cellulase of fungal origin, the metabolite was no longer detectable, either in the medium or in the cells. Calcium deprivation in the medium and treatment with the elicitors MeJ and cellulase lowered the cellular level of all the sterols tested. The possible role of 1α,25(OH)2D3 is discussed in light of these results.