A significant contribution of the forestomachs in net calcium (Ca2+) absorption from the gastrointestinal tract has been postulated from in vivo and in vitro studies in different ruminant species. However, the potential role of vitamin D3 and its metabolites in controlling these mechanisms is still under discussion. It was therefore the aim of the present study to investigate the effectiveness of treatment with vitamin D3 in stimulating active Ca2+ absorption from sheep rumen. Four mature, non-lactating, non-pregnant sheep that had been treated 7 and 4 days before the Ca2+ flux rate measurements with intramuscular injections of 300000 IU of vitamin D3 each in aqueous solution were used. Two female and three male placebo-treated sheep served as controls. To characterize the effects of vitamin D3 application on plasma parameters the time courses of total calcium, inorganic phosphate, calcitriol and intact parathyroid hormone (iPTH) were recorded. In vitro studies of unidirectional Ca2+ flux rates across isolated, intact rumen wall epithelia were carried out by applying the Ussing-chamber technique. Western blot analysis and reverse transcriptase-polymerase chain reaction analysis (RT-PCR) were applied to identify vitamin D receptors (VDR) in ruminal and jejunal tissues. In addition, Western blot analysis for qualitative examination of epithelial calbindin D9k levels was carried out in these tissues. Total calcium and phosphate levels in plasma were not significantly affected treatment with vitamin whereas calcitriol concentrations significantly increased by about 130 and 63% after the first and second application, respectively. In contrast, iPTH tended to decrease by about 60% indicating regulatory effects of calcitriol on systemic Ca homeostasis. The Ca2+ flux rate measurements in Ussing-chambers revealed significant net Ca2+ absorption indicating the contribution of active mechanisms for Ca2+ transport in rumen epithelia. This, however, was not significantly affected by increased calcitriol concentrations in plasma. Western blot analysis on the basis of a human recombinant VDR protein and RT-PCR clearly indicated the presence of VDR in ruminal and jejunal epithelia, but, in contrast to jejunum, this was not reflected by respective amounts of calbindin-D9k in ruminal tissues. The results suggest the absence of classical calbindin-D9k-mediated mechanisms for active Ca2+ transport in sheep rumen.