In mammals, the meiosis division in testes produces equal numbers of two different types of gametes: X chromosome-bearing sperm (X-spermatozoa) and Y chromosomebearing sperm (Y-spermatozoa), which have equal potential to fertilize the oocytes. Therefore, the expected 1: 1 sex ratio is observed. However, under some conditions like endocrine disruptors (EDs) exposure the sex ratio is deviated than the expected with more males or more females. And recently many hypotheses have been postulated to explain the mechanism of sex ratio deviation; however none of them introduced a proven experimental explanation. To solve this enigma, we hypothesized that the differences between X- and Y-spermatozoa survivability under specific conditions due to differences in their chromosome contents are the key leading to the sex ratio alteration. To examine our hypothesis, we combined two techniques; first, hypo-osmotic swelling (HOS) test that was applied to test viability of spermatozoa and second, fluorescence in situ hybridization that was applied on HOS-treated spermatozoa to define sex chromosome composition. In the present study, human spermatozoa were incubated with a group of EDs represent a widespread chemicals in the environment bisphenol A (BPA, 100 μM), nonylphenol (NP, 10 μg/ml), 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD, 2.5 μg/ml), genistein (Gen, 100 μM), and the following pesticides, dibromochloropropane (DBCP, 10 μg/ml), atrazine (Atraz, 500 μM), and diazinone (Diaz, 500 μM) for 6 hr at 37℃ in 5% CO2. Then, the viability of spermatozoa and their sex chromosome contents were evaluated simultaneously. Among seven chemicals studied only four chemicals (Atraz, DBCP, TCDD, and Diaz) significantly decreased Y-sperm viability when compared to those of X-spermatozoa in the same treatment group and viability of Y-spermatozoa when compared to those in the negative and positive (DMSO) control groups (p<0.05). Also, in these four treatment groups the sex ratio of live sperm population was significantly lowered compared to the control groups (p<0.05). Otherwise, Gen, BPA, and NP did not show any significant effect on viability of Yspermatozoa or decreasing sex ratio in live sperm population as compared to the control groups. It has been proven that TCDD, DBCP, and the pesticides decrease the sex ratio, but the same effect was not observed in case of Gen, BPA, and NP. From the present findings, there is no doubt that the EDs may alter sex ratio via decreasing Y-spermatozoa viability.