These experiments were carried out to develop new techniques for In Vitro separatin of X-and Y-bearing spermatozoa. The bull semen was applied to the various Gel-Columns filled with swellen Sephadex G-50 Fine and then elutriated wtih Locke solution (elutriation rate; 1ml/3-4min., 1ml/1-2min.). Elutriated solution was fractionated into 1ml by automatic Fraction Collector and spermatozoa included in each fraction were subjected to the estimation of viability and recovery rate, and to B-body test. The results obtained in these experiments were summarized as follows: 1. When the column size and the elutriation rate were adjusted to 15$ imes$1.6cm and 1ml/3-4min., respectively, the highest sperm concentration was obtained from the 8th to the 12th fraction. 2. As a trend, the viability of spermatozoa was improved by chromatography, and the degree of improvement ranged 5 to 10 percentage. 3. The average recovery rate of spermatozoa applied to column was 73.2 percentage and ranged 52.6 to 81.3 percentage. 4. The lowest rate of B-body bearing spermatozoa following chromatography was obtained when the column size and the elutriation rate were adjusted to 15$ imes$0.8cm and 1ml/1-2min., respectively.