Abstract: The preparation of milligram quantities of three vitamin D₃ isomers, pre-vitamin D₃, lumisterol₃ and tachysterol₃, were carried out in a laboratory scale first by irra-diating 7- dehydrocholesterol in a phototherapy chamber equipped with UVB lamps, fol-lowed by using a two- step high performance liquid chromatography (HPLC) with asemi - preparative normal phase column and an analytical reverse- phase column. The finalproducts obtained were identified by UV spectrophotometry and HPLC. According to thedetection limits for the three isomers, no contamination with any other isomers was de-tected in the previtamin D₃, lumisterol₃ and tachysterol₃ preparations, except that a verysmall amount of vitamin D₃, constituting no more than 0.25% of the product, was foundin previtamin D₃ preparation.