Posttranslational processing of progastrin to a carboxy terminally amidated form (G-NH₂) is essential for its effect on gastric acid secretion and other biological effects mediated by gastrin/CCK-B receptors. The immediate biosynthetic precursor of G-NH₂, glycine-extended gastrin (G-Gly), does not stimulate gastric acid secretion at physiological concentrations but is found in high concentrations during development. G-NH₂ and G-Gly have potent growth stimulatory effects on gastrointestinal tissues, and G-NH₂ can stimulate proliferation of human kidney cells. Thus we sought to explore the actions of G-NH₂ and G-Gly on the human embryonic kidney cell line HEK 293. HEK 293 cells showed specific binding sites for ¹²⁵I-labeled Leu¹⁵-G17-NH₂ and ¹²⁵I-Leu¹⁵-G₂₁₇-Gly. Both G-NH₂ and G-Gly induced a dose-dependent increase in [³H]thymidine incorporation, and both peptides together significantly increased [³H]thymidine incorporation above the level of either peptide alone. G-NH₂ and G-Gly were detected by radioimmunoassay in serum-free conditioned media. Antibodies directed against G-NH₂ and G-Gly lead to a significant reduction in [³H]thymidine incorporation. G-NH₂ but not G-Gly increased intracellular Ca²⁺ concentration. We conclude that G-NH₂ and G-Gly act cooperatively via distinct receptors to stimulate the growth of a nongastrointestinal cell line (HEK 293) in an autocrine fashion.