The method and its reliability for sex determination by examining X-chromatin in the nucleus of hair root cell and buccal mucosa cell are presented: 1) X-chromatin in the nucleus of hair cortex cell was stained by Feulgen reaction after bleaching melanin granules and the frequency of X-chromatin was calculated under a ordinary microscope. An another method, fluorescent Feulgen reaction with acriflavine using a fluorescence microscope, was attempted. The results obtained from both methods indicated significantly higher values in the female samples than those in the male ones. 2) The other hand, the frequency of Y-chromatin was determined after quinacrine staining using a fluorescence microscope, and was also distinguishable between the male and female samples. 3) Using a single specimen, sex determination from the frequency of X- or Y-chromatin was practicable through combined treatment of quinacrine staining and fluorescent Feulgen reaction. 4) These methods were available during 16 to 64 weeks at least regarding samples kept dry. It is concluded that sex determination from frequency of sex chromatin after treatment described above is reliable.