Vitamin D2 is less toxic in rats when compared to vitamin D3. Our laboratory has been involved in research projects which were directed towards identifying the possible mechanisms responsible for the toxicity differences between vitamins D2 and D3 in rats. The present research project was designed to isolate and identify new metabolites of vitamin D2 from serum of rats which were fed toxic doses of vitamin D2. Hypervitaminosis D2 was induced in 30 rats by feeding each rat with 1000 nmol of vitamin D2/day x 14 days. The rats were sacrificed on the 15th day and obtained 180 ml of serum. The lipid extract of the serum was directly analyzed by a straight phase HPLC system. The various vitamin D2 metabolites were monitored by their ultraviolet (UV) absorbance at 254 nm. One of the UV absorbing peaks did not comigrate with any of the known vitamin D2 metabolites. This unknown metabolite peak was further purified by HPLC and was then subjected to UV absorption spectrophotometry and mass spectrometry. The structure assignment of the new metabolite was established to be 4,25-dihydroxyvitamin D2 [4,25(OH)2D2] by the techniques of UV absorption spectrophotometry and mass spectrometry and by the new metabolite's susceptibility to sodium metaperiodate oxidation. At present the biological activity of this unique 'A-ring' hydroxylated vitamin D2 metabolite is not known. As this new metabolite is isolated from the serum of rats intoxicated with vitamin D2, we speculate that 4,25(OH)2D2 may be playing an important role in the deactivation of vitamin D2.