To determine new polymorphisms in the antioxidant enzymes superoxide dismutase, glutathione peroxidases, catalase, and microsomal glutathione transferase 1, a search of the human expressed sequence tags (EST) database was performed (with BLAST 2.0). When any mutation, indicated by the BLAST search, gave rise to a nonconservative amino acid change we performed polymerase chain reaction (PCR) restriction analysis and/or sequence analysis of genomic DNA from human subjects in order to verify these potential polymorphisms. Of nine indicated polymorphisms from the EST analysis found in four different antioxidant enzymes, we could verify one, an amino acid substitution Pro-Leu at amino acid position 197 (P197L), in the glutathione peroxidase 1 gene. The corresponding allele frequencies were approximately 70/30%. In addition, a silent mutation (1167T/C) in the catalase gene indicated by the BLAST search could also be verified. Six to nine individuals were analyzed per indicated polymorphism, so that only common polymorphisms would be found. The indicated mutations not verified by direct analysis thus cannot be excluded as allelic variation in the human population. These results show that the EST database can be used to search for polymorphisms in genes with high abundance in the human EST database. In addition to the EST analysis, PCR/single-strand conformation polymorphism (SSCP) was employed for the analysis of the microsomal glutathione transferase 1 gene. No polymorphism in the coding sequence could be detected in the gene by either method. The high degree of conservation of the microsomal glutathione transferase 1 gene indicates an important physiological function for this enzyme.