Recently, 1,25-dihydroxyvitamin D3 (1,25-D3) and less hypercalcemic analogs were shown to exert a delayed cytotoxic effect on rat C6 glioma cells. 1,25-D3 induces in these cells a programmed cell death, accompanied by the induction of c-myc, p53 and gadd 45 genes. The involvement of the intracellular vitamin D receptor (VDR) remained to be determined. In this lethal process, we have investigated its role in a subclone of C6 cells, which was isolated on the basis of its resistance to 1,25-D3, and in which VDR expression was not detected either at the mRNA or protein levels. The stable transfection of a rat VDR cDNA into this clone restored its susceptibility to the cytotoxic effects of 1,25-D3. This phenomenon was accompanied by a dramatic upregulation of c-myc mRNA expression, as already described in a C6-sensitive clone. These results provide the first evidence that VDR expression, if not sufficient, is necessary to mediate 1,25-D3 cytotoxic effect in C6 glioma cells. Since VDR mRNA expression has been already reported in human brain tumors, our data imply that the identification of VDR expression could become a prerequisite in any strategy of glioma treatment with vitamin D analogs.