The structure of full-length rat prodynorphin cDNA and the corresponding gene has been determined. The 2400 base rat prodynorphin mRNA is encoded by four exons. Exons 1 and 2 encode the majority of the 5'-untranslated sequence, while exons 3 and 4 contain the translated region; the entire 3' -untranslated region is contained on exon 4 as well. RNase protection studies, in which a genomic DNA fragment was used to generate a cRNA hybridization probe, have determined the major transcriptional initiation site for both brain and testicular prodynorphin mRNA. Transient expression of transfected fusion genes containing the 5'-flanking DNA of the rat prodynorphin gene linked to the structural sequence of a reporter gene has been used to identify specific genomic DNA fragments from the prodynorphin gene locus which are capable of acting as transcriptional promoters. Multiple regions of genomic DNA appear to have transcriptional promoter activity when introduced into various eukaryotic cell lines.