The biosynthesis of gastrin involves a complex series of post-translational processing reactions that result in the formation of a biologically active secretory product. To study the mechanisms for two specific reactions in gastrin processing, namely dibasic cleavage and amidation, we infected AtT-20, GH3, and Rin5-f cells with the retroviral expression vector, pZip-NeoSV(X), containing human gastrin cDNA. We detected gastrin and its glycine extended post-translational processing intermediates (G-gly) in the media and cell extracts of successfully infected cells. Characterization of the molecular forms of gastrin in these cell lines revealed that GH3 and Rin5-f processed gastrin in a manner similar to antral G-cells but the cleavage of the Lys74-Lys75 bond that converts G34 to G17 appeared to be suppressed in AtT-20 cells. Even after conversion of this site to Arg74-Arg75 via site-directed mutagenesis, the At-20 cells synthesized G34 predominantly. All of the infected cells amidated gastrin but the gastrin/G-gly ratio, a reflection of amidation within the cells, was enhanced in GH3 and Rin5-f cells but diminished in AtT-20 cells upon treatment with dexamethasone (10(-4) M) for 3 days. The dibasic cleavage of gastrin was uneffected by dexamethasone. Our data suggest that the activities of post-translational processing reactions responsible for the synthesis of biologically active gastrin exhibit considerable tissue and substrate specificity.