Growth factor effects of precursor forms of gastrins have become evident in recent years. However, intracellular pathways that mediate growth effects of the precursor molecules are not known. In previous studies, we reported an increase in Tyr phosphorylation of pp60^c-Src in intestinal epithelial cells (IEC) in response to the fully processed form of gastrin [gastrin_1–17 (G17)]. We have now examined whether c-Src kinase is similarly phosphorylated and activated in response to the full-length precursor molecule, progastrin (PG)_1–80, (recombinant human PG) in IEC cells. We found a significant increase in pp60^c-Src kinase activity in response to both G17 and PG (0.1–1.0 nm), suggesting that growth effects of both the precursor and fully processed gastrin molecules may be mediated via similar pathways. On the other hand, pp62^c-Yes was not phosphorylated or activated in response to either G17 or PG. To examine whether c-Src kinase mediates proliferative effects of PG, IEC cells were microinjected with anti-Src-IgG and ³H-thymidine (³H-Tdr) uptake of the cells measured. Control cells received nonimmune IgG. The ³H-Tdr uptake of cells stimulated with 1.0 nm PG was significantly reduced in cells microinjected with anti-c-Src-IgG; control IgG had no effect. In cells stimulated with 1.0% fetal calf serum, microinjection with c-Src-IgG had no effect on ³H-Tdr uptake. The specificity of the effect was further confirmed by blocking the inhibitory effect of anti-c-Src-IgG with antigenic Src peptide. These results suggest that activation of c-Src kinase likely represents a critical step in mediating proliferative effects of both the precursor and fully processed forms of gastrins on IEC.