Abstract: To determine the frequency of hepatitis B, hepatitis C, Human Immunodeficiency Virus (HIV) and syphilis infections in Iranian blood donors. The prevalence of serological markers of hepatitis B, hepatitis C, HIV and syphilis infections were evaluated in 318029 consecutive volunteer blood donors attending to Tehran blood transfusion service from March 2005 to March 2006. Those positive for hepatitis B surface antigen, anti-HCV, anti-HIV1/2 and VDRL (venereal disease research laboratory) reactivity were analyzed with a second independent HBsAg enzyme immunoassay (EIA) and neutralization assay; an additional independent anti-HCV EIA and HCV-RIBA assay; second independent anti-HIV1/2 test, HIV western blot and fluorescent Treponemal Antibody Absorbed (FTA-ABS), respectively. In 318029 participants, prevalence of positive HBsAg, HCV RNA, HIV western blot and FTA-ABS was 1684 (0.487%), 323 (0.093%), 11 (0.003%) and 19 (0.005%), respectively. In 1014 subjects randomly selected from these 318029 participants, besides standard interview, physical exam and routine serologic tests; anthropometric and biochemical were studies. In this selected group frequency of HBsAg was 3 (0.29, 95% CI: 0-0.64%); frequency of anti-HCV was 21 (2.07%), but it was (0.09%, 95% CI: 0-0.30%) by confirmatory HCV RNA test; frequency of HIV-Ab1, 2 was 8 (0.78%), but it was 2 (0.19%, 95% CI: 0-0.48%) by confirmatory test; frequency of RPR was 0 (0%, 95% CI: 0-0.30%). Despite excluding subjects with high-risk behaviors by standard interview and physical examination, still a few asymptomatic hepatitis B, hepatitis C, HIV-infected subjects existed among volunteer blood donors with demographic and biochemical findings similar to non-infected ones.