Abstract A recombinant plasmid containing the rat preprodynorphin cDNA was introduced into the rat anterior pituitary cell line, GH4C1. These cells normally express growth hormone and prolactin but not prodynorphin. Stable transformants were isolated and analyzed for the expression and processing of prodynorphin. Chromatographic analyses demonstrated that the prodynorphin was incompletely processed in GH4C1 cells. Analyses of the peptides by specific radioimmunoassays to chemically synthesized peptides showed that the cells have the ability to process both at dibasic and monobasic cleavage sites. The release of prodynorphin-derived peptides paralleled that of prolactin upon stimulation with thyrotropin-releasing hormone, forskolin or carbachol suggesting that the prodynorphin-derived peptides and prolactin are sequestered in similar physiologically responsive compartments. These data suggest that the GH4C1 cells incompletely process prodynorphin. The processing in GH4C1 cells occurs both at monobasic and dibasic cleavage sites.