The inhibition of glucose-6-phosphate dehydrogenase (G6PD) expression by arachidonic acid occurs by changes in the rate of pre-mRNA splicing. Here, we have identified a cis-acting RNA element required for regulated splicing of G6PD mRNA. Using transfection of G6PD RNA reporter constructs into rat hepatocytes, the cis-acting RNA element involved in this regulation was localized to nucleotides 43-72 of exon 12 in the G6PD mRNA. In in vitro splicing assays, RNA substrates containing exon 12 were not spliced. In contrast, RNA substrates containing other regions (exons 8-9 or exons 10-11) of the G6PD mRNA were efficiently spliced. Furthermore, exon 12 can inhibit splicing when substituted for other exons in RNA substrates that are readily spliced. This activity of the exon 12 regulatory element suggests that it is an exonic splicing silencer (ESS). Consistent with its activity as a splicing silencer, spliceosome assembly was inhibited on RNA substrates containing exon 12 compared to RNAs representing other regions of the G6PD transcript. Elimination of nucleotides 43-72 of exon 12 did not restore splicing of exon 12 containing RNA, thus the 30 nt element may not be exclusively a silencer. The binding of hnRNPs K, L, and A2/B1 from both HeLa and hepatocyte nuclear extracts to the element further supports its activity as a silencer. In addition, SR proteins bind to the element consistent with the presence of enhancer activity within this sequence. Thus, an ESS is involved in the inhibition of splicing of a constitutively spliced exon in the G6PD mRNA.