A significant chromatographic isotope effect is reported for 1,25-dihydroxyvitamin D3 in a wide variety of HPLC separation systems. The effect is also observed for 24,25-dihydroxyvitamin D3. Retention times differ from less than 1% up to 4% depending on the separation system and the degree and position of tritium substitution. Such an effect must be corrected for whenever both labeled and unlabeled vitamin D metabolites are used in HPLC cochromatography or assay recovery studies.