The primary structure of the human microsomal glutathione S-transferase gene (GST12) was determined by genomic cloning. The gene structure of GST12 spans 12.8 kb and consists of four exons and three introns. The coding sequence resides on exons 2, 3, and 4. Sequencing of the exons revealed two nucleotide differences compared to a previous report of the cDNA sequence. The substitutions, however, were silent, as they did not alter amino acid composition or restriction enzyme sites. All introns commenced with nucleotides GTAA at the 5' boundary and ended with nucleotides AG at the 3' boundary, in agreement with the proposed consensus sequence for intron spliced donor and acceptance sites. The presence of an in-phase stop codon and an upstream false start codon in the 5'-untranslated region was confirmed. Although it was previously predicted that there existed another start codon in-phase and within 50 bp of this stop codon, coding for a second mini-cistron, we could not identify another start codon for greater than 200 bp prior to the stop codon. Thus, initiation is suppressed at the first or false start codon due to either the closeness of the stop codon or the suboptimal context of the codon.