Understanding tissue vitamin D pools is important with respect to normal physiology and has relevance to its role in cancer prevention. In the context of breast cancer, the expression of VDR, CYP27B1 and CYP24A1 in normal breast supports the concept that vitamin D steroids accumulate in this tissue and that reduced availability of D3 metabolites during deficiency contributes to tumor promotion. However, no studies have measured accumulation of D3 or 25D3 in breast tissue as a function of diet or in relation to other body pools. We are addressing whether D3 or its metabolites accumulate in mammary gland and whether dietary deficiency or genetic ablation of vitamin D Binding Protein (DBP) alters tissue pools. SNPs in the DBP gene are known modifiers of vitamin D status as DBP transports D, 25D and 1,25D and facilitates uptake in some tissues. In our studies, WT and DBP knockout (DBPKO) mice were fed AIN93 diets with 1000IU/kg D3 (CON) for 8 wks at which time half were subjected to D3 deficiency (0IU D3/kg diet; DEF) while the remaining cohorts continued on CON diets. After 4 wks, mammary gland and liver were analyzed by LC/MS/MS for D3, D2, 25D3 and 25D2. In WT mice on CON diets, D3 and 25D3 accumulation in mammary gland was comparable to that in liver. Comparison of WT vs DBPKO mice indicated that: 1) on CON diet, D3 (but not 25D3) accumulation in mammary gland was higher in DBPKO mice relative to WT mice; 2) on CON diet, higher levels of both D3 and 25D3 were detected in mammary gland of DBPKO mice relative to liver [as opposed to comparable levels in these two tissues of WT mice]; 3) DEF diets reduced D3 and 25D3 content of mammary gland or liver similarly in WT and DBPKO mice. In summary, these are the first data to demonstrate that D3 and 25D3 accumulate in mammary gland and that both steroids can be released during vitamin D deficiency. Furthermore, DBP is not required for release of D3 or 25D3 from tissues during dietary deficiency. Xenograft studies confirm that 25D3 accumulation in breast tumors also correlates with dietary intake and serum 25D3. The implication of this data is that vitamin D activity in mammary gland and breast cancers is highly likely to reflect vitamin D intake.