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This study aims to determine the most frequent titers of anti-A and anti-B (both presumed immunoglobulin [Ig]M and IgG) in Iranian group O blood donors and to compare these titer values with those found in other studies. In addition, alloantibody production and plasma levels of four IgG subclasses were compared between the high-titer and non-high-titer study groups. This study investigated anti-A and anti-B titers in 358 plasma samples. Based on these results, two study groups (high-titer and non-high-titer) were formed. Antibody detection tests were performed to detect unexpected antibodies to D, C, c, E, e, K, k, Fya, Fyb, Jka, Jkb, M, N, S, s, P1, Lea, and Leb. Four IgG subclasses were also evaluated through nephelometry assay. The most frequent titer obtained by room temperature and indirect antiglobulin tube tests was 256. The frequency of titers ≥512 was 31.5 percent. None of the cases showed unexpected RBC alloantibodies. IgG2 levels were significantly higher in the high-titer group. Evaluation of isoagglutinin titers in group O Iranian blood donors can provide insight into the frequency of isoagglutinin titers both within the Iranian population and as compared with other populations. A significant difference in IgG2 levels between the high-titer and non-high-titer groups was identified. More investigation needs to be conducted on the root cause of this finding. Immunohematology 2021;37:5-12 . This study aims to determine the most frequent titers of anti-A and anti-B (both presumed immunoglobulin [Ig]M and IgG) in Iranian group O blood donors and to compare these titer values with those found in other studies. In addition, alloantibody production and plasma levels of four IgG subclasses were compared between the high-titer and non–high-titer study groups. This study investigated anti-A and anti-B titers in 358 plasma samples. Based on these results, two study groups (high-titer and non–high-titer) were formed. Antibody detection tests were performed to detect unexpected antibodies to D, C, c, E, e, K, k, Fya, Fyb, Jka, Jkb, M, N, S, s, P1, Lea, and Leb. Four IgG subclasses were also evaluated through nephelometry assay. The most frequent titer obtained by room temperature and indirect antiglobulin tube tests was 256. The frequency of titers ≥512 was 31.5 percent. None of the cases showed unexpected RBC alloantibodies. IgG2 levels were significantly higher in the high-titer group. Evaluation of isoagglutinin titers in group O Iranian blood donors can provide insight into the frequency of isoagglutinin titers both within the Iranian population and as compared with other populations. A significant difference in IgG2 levels between the high-titer and non–high-titer groups was identified. More investigation needs to be conducted on the root cause of this finding. Immunohematology 2021;37:5–12 .