Complementary DNAs encoding the hamster type 2 3 beta-hydroxysteroid dehydrogenase/delta 5-->4 isomerase were isolated from liver and kidney cDNA libraries. Nine clones were isolated containing identical coding and 3' untranslated sequences. However, six of the clones contained a 68-nucleotide stretch in the 5' untranslated region that was missing in the other three clones. Primers were designed to flank this region and the polymerase chain reaction (PCR) was performed on hamster liver and adrenal RNA. Two PCR products were amplified of the predicted molecular sizes and with the expected sequence. Primers were then designed to amplify sequences encompassing this region from hamster genomic DNA. Sequencing of the resultant PCR products demonstrated that the 68-nucleotide stretch missing in some transcripts corresponded exactly to the second of three exons identified. We conclude that the 5' untranslated region of this mRNA is transcribed from at least three exons, and that the sequence of the second of these exons is spliced out of some of the RNA transcripts.