Abstract Serologic ABO blood typing is routinely performed using anti-A and anti-B sera to distinguish four phenotypes (A, B, AB, and O). Restriction fragment length polymorphisms (RFLPs) and DNA sequence studies offer the possibility of direct ABO genotyping. We used polymerase chain reaction-RFLP analysis to determine the frequency of O¹ and O² alleles in 82 unrelated blood donors in São Paulo, Brazil, known to be group O. Genomic DNA was extracted from blood leukocytes by a modified salting-out method. Different genotypes (O¹O¹, O¹O², O²O²) were identified after digestion with restriction enzymes KpnI, HpaII, and AluI, followed by agarose gel electrophoresis. Of 82 samples analyzed, 74 were O¹O¹ , 7 were O¹O² , and 1 was O²O² . These results showed the frequency of O¹O¹, O¹O² , and O²O² genotypes to be 90.24 percent, 8.53 percent, and 1.22 percent, respectively, in blood donors in São Paulo, Brazil. Immunohematology 2001;17:111–116.