Previous methods of enriching sperm with a higher percentage of Y-bearing sperm have been questioned because the claims that Y enrichment was present were based on quinacrine staining of the Y chromosome, and the enrichment was not confirmed by polymerase chain reaction (PCR) or fluorescent in situ hybridization (FISH) techniques. A technique was evaluated that theoretically could increase the percentage of X-bearing sperm by isolating a fraction of the "heaviest" sperm by passing them through 12 layers of discontinuous Percoll gradient. Initially 12 specimens were checked both before and then after separation with 12 layers of Percoll for percentage of Y sperm. The median for baseline Y percentage was 49% and after processing the percentage of Y dropped to 10%. An additional 19 specimens were checked after separation only. The median was 19%. The sample with the lowest preseparation % of quinacrine staining sperm was 45% and the highest was 54%. After 12-layer Percoll, the lowest percentage was 3% and the highest was 24%. There have been claims that quinacrine staining can falsely increase apparent Y-bearing sperm enrichment following certain separation procedures, e.g.. albumin separation, by nonspecific staining of autosomal chromosomes. If anything, then, it should falsely decrease X-bearing sperm enrichment. Thus, 12-layer Percoll separation may actually enrich for X-bearing sperm or possibly this procedure somehow nonspecifically inhibits the ability of quinacrine to stain the Y chromosome.