The objective was to examine the separation of X- and Y-bearing spermatozoa in a modified swim-up procedure using fluorescent in situ hybridization (FISH), and to find out the influence of swim-up time on the ratio of X- and Y-bearing spermatozoa.Prospective study.Reproductive testing laboratory in a university hospital.Normal spermatozoa samples were obtained from 10 volunteers by masturbation after sexual abstinence for 3-5 days.Spermatozoa were put into 18 tubes with 0.25 ml in each, then mixed with HTF medium and centrifuged for 5 min (400 x g). The supernatant was removed and discarded and 0.5 ml HTF was added slowly along the tube wall. Motile spermatozoa were collected after swimming up in different times (from 5 up to 150 min, with a total of 17 intervals). The X- and Y-bearing spermatozoa were determined using the FISH technique. The X/Y dual-color CEP probes that were marked by fluorescein isothiocyanate (FITC) and Texas red were applied to analyze the ratio of X- and Y-bearing spermatozoa. The FISH staining slides were analyzed under an immunofluorescence microscope. About 1000-1500 spermatozoa were counted per slide.The percentages of X- and Y-bearing spermatozoa were calculated.The study results suggested that the total ratio of hybridization was 98.33%. The ratio of X-bearing spermatozoa after swimming up for different amounts of time is 50.03 +/- 0.91% at 0 min, 50.45 +/- 2.06% after 15 min, 50.61 +/- 2.47% after 30 min, 50.16 +/- 2.67% after 60 min, 50.72 +/- 2.64% after 90 min, and 50.56 +/- 2.20% after 150 min. The statistical analysis showed that there were no significant differences among different swim-up times in the ratio of X-bearing spermatozoa.There was no significant effect of swim-up time on the ratios of X- and Y-bearing spermatozoa using a modified swim-up procedure. No direct evidence was found that the swim-up procedure for separating motile spermatozoa to use for either intrauterine insemination (IUI) or in vitro fertilization (IVF) would lead to an imbalance of boys and girls.