Analysis of the expressed sequence tag (EST) database by sequence alignment allows a rapid screen for polymorphisms in proteins of physiological interest. The human zeta class glutathione transferaseGSTZ1has recently been characterized and analysis of expressed sequence tag clones suggested that this gene may be polymorphic. This report identifies threeGSTZ1alleles resulting from A to G transitions at nucleotides 94 and 124 of the coding region,GSTZ1*A – A94A124;GSTZ1*B – A94G124;GSTZ1*C – G94G124. Polymerase chain reaction/restriction fragment length polymorphism analysis of a control Caucasian population (n = 141) showed that all three alleles were present, with frequencies of 0.09, 0.28 and 0.63 forZ1*A,Z1*BandZ1*C, respectively. These nucleotide substitutions are non-synonymous, with A to G at positions 94 and 124 encoding Lys32to Glu and Arg42to Gly substitutions, respectively. The variant proteins were expressed inEscherichia colias 6X His-tagged proteins and purified by Ni-agarose column chromatography. Examination of the activities of recombinant proteins revealed that GSTZ1a–1a displayed differences in activity towards several substrates compared with GSTZ1b–1b and GSTZ1c–1c, including 3.6-fold higher activity towards dichloroacetate. This report demonstrates the discovery of a functional polymorphism by analysis of the EST database.