Introduction: Reactive oxygen Species (ROS) are a group of free radicals that in excessive amounts showed to have negative influence on sperm quality and function. Some Clinical entities such as varicocele, spinal cord injury and genital infections showed to be associated with high ROS levels in semen.In this study we assessed ROS levels in seminal plasma of infertile men and compare with its level in healthy donors. We also determined the ROS level in semen of infertile patients according to the etiology of infertility, and also the effect of smoking on its level. Materials and Methods: We selected 95 infertile patients and 63 healthy donors as control. Complete physical examination, semen analysis, scrotal sonography and hormone assay were done for all patients. Azoospermic patients were excluded from the study. ROS level in semen was measured by chemiluminescence assay in both groups. Patients also divided in two groups according to the etiology of infertility. Results: We were excluded 32 infertile patients from the study. Semen specimens from 126 individuals, 63 samples from infertile men and 63 from healthy controls were studied. The mean age of normal subjects and infertile men were 30.78±3.73 years and 31.43±6.60 years respectively. The mean Abstract of the 8th Royan International Twin Congress, Tehran, Iran, 5-7 September 2007 18 Yakhteh Medical Journal, Vol 9, Sup 1, Summer 2007 ROS level in normal men was 180.05 RLU, and 1852.04 RLU in infertile patients, which is significantly higher in case group (p=0.000). Fifty patients had varicocele and for the other 13 patients no any specific etiology was found. The mean ROS level in varicocele patients was 2215.42 and in unknown group was 454.44 (p=0.048). Conclusion: Our study showed that level of ROS in seminal fluid of infertile men is significantly higher than fertile donors and also in infertile patients with varicocele is higher than patients with unknown cause.