The reaction of 2,3-diaminonaphthalene (DAN) with Se(IV) to form a fluorescent Se-DAN complex is the basis of a fluorometric method for determination of Se. With the aid of metabolically incorporated ⁷⁵Se the method was critically re-examined. The study showed that loss of ⁷⁵Se was negligible when liver or blood was microwave-dried or thermally dried at temperatures up to 120°C; during HC1 reduction of Se(VI) to Se(IV), a temperature up to 210°C could be used with no loss of ⁷⁵Se; it was unnecessary to perform pH adjustment of solution for formation of Se-DAN complex before solvent extraction; it was unnecessary to carry out the chelation/extraction step in diffuse light; solvent phase containing Se-DAN complex could be left in contact with aqueous phase up to one week under fluorescent light with no effect on analytical results. From this study we were able to dispute or overcome a number of criticisms and myths which have been laid against the fluorometric method for many years. As a result, an improved single tube method was developed. The withinbatch variation of the improved method was about 2%, while the between-batch variation over a period of 2 years was less than 10%. The method can handle 200 samples per batch and is applicable to a wide range of biological samples including liver, orchard leaves, barley, wheat, lucerne (alfalfa), poultry feed, fish, hair, blood, urine, and milk.