Previous studies showed that the expression of an mRNA corresponding to VDUP-1 was decreased within MNU-induced rat mammary tumors. RNA from mammary tissue was DNase treated and reverse transcribed and the resulting cDNA was amplified using primers designed to amplify VDUP-1 (382 bp fragment) and glyceraldehyde-6-phosphate dehydrogenase (GAPDH) (416 bp fragment). Analysis of mammary cDNA derived from untreated or MNU-treated rats indicated that VDUP-1 expression within tumor tissue was significantly decreased, a finding which agrees with previous Northern blotting experiments. The differential expression was confirmed in tissue sections using an antisense VDUP-1 riboprobe for in situ hybridization studies which demonstrated that VDUP-1 staining in all cell types within tumor tissue was greatly decreased. VDUP-1 mRNA was expressed to a greater extent within epithelial cells and to a much lesser extent within stromal cells, including endothelial cells, in untreated mammary tissue. A significant decrease in VDUP-1 expression was detected as early as six weeks after MNU treatment, before tumors had formed. Bilateral ovariectomy did not alter VDUP-1 expression in untreated mammary tissue and ovariectomy prior to MNU treatment prevented tumor formation, as well as the associated decrease in VDUP-1 expression. The relative expression of VDUP-1 was higher in lung tissue than in adrenal, heart, kidney, liver, mammary, muscle, and ovary. Treatment of a cell line derived from an MNU-induced rat mammary tumor (MNU cells) with 1,25-dihydroxyvitamin D3 resulted in a significant increase in VDUP-1 expression and also inhibited cell growth in the absence of serum. The data are consistent with a role for VDUP-1 in mediating the inhibitory effects of 1,25-dihydroxyvitamin D3 on tumor cell growth.