Previous work on preimplantation genetic diagnosis (PGD) of single gene disorders by the first polar body (IPB) analysis has demonstrated that the genotype of a considerable number of embryos resulting from heterozygous oocytes cannot be predicted without testing their second PB (IIPB). To overcome this limitation we introduce a two-step DNA analysis of oocytes using both IPB and IIPB to identify hemizygous mutation-free oocytes following the second meiotic division. In the application of the approach to PGD of cystic fibrosis (CF) Delta F-508 mutation, sickle cell disease, and hemophilia B, 80 oocytes were studied by both PBs, resulting in the identification and transfer of 32 homozygous normal embryos. A follow-up genotyping of 52 embryos, resulting from oocytes tested by both IPB and IIPB demonstrated the accuracy of the predicted genotypes. In addition to a nested PCR analysis of the mutant genes in PBs and resulting embryos, simultaneous amplification of different polymorphic markers was performed, demonstrating the reliability of the two-step polar body analysis of oocytes.