Fluorescent in situ hybridization of decondensed sperm cells was used to evaluate the effectiveness of separating X- and Y chromosome bearing sperm cells using a combination of Percoll and NycoPrep media. Semen samples from seven healthy men were divided into three equal aliquots. One aliquot served as an untreated control, the other two were either applied to a seven-step Percoll gradient (100%-40%), or to a seven-step Percoll gradient (100%-40%) underlayered with 100% NycoPrep for sperm purification. After centrifugation Y-bearing sperm cells in the pellet and in the control sample was determined by fluorescent in situ hybridization. A significantly reduction of Y-bearing sperm cells were observed in the pellet of the Percoll + NycoPrep centrifugation (39.3% Y-bearing sperm cells) compared to the control sample and to Percoll centrifugation without NycoPrep. The Percoll centrifugation without NycoPrep did not show a significant reduction of Y-bearing sperm cells in the pellet compared to the control sample. By the addition of a medium with a density higher than 100% Percoll a significantly increased separation of X and Y-bearing sperm cells can be achieved. This study opens new perspectives for improving separation of X- and Y-bearing spermatozoa by simple centrifugations. The described centrifugation technique can be applied in connection with preimplantation diagnosis in order to create more pre-embryos of the desired gender.