Human pancreatic ductal adenocarcinomas overexpress the epidermal growth factor (EGF) receptor. Betacellulin is a mitogenic polypeptide that binds and activates this receptor. To determine whether betacellulin has a role in human pancreatic cancer, we studied its expression in cultured human pancreatic cancer cell lines and in normal and cancerous pancreatic tissues. Five of 6 pancreatic cancer cell lines expressed the 3 kb betacellulin mRNA moiety, T3M4, MiaPaCa-2 and COLO-357 cells exhibiting the highest expression levels. EGF, heparin-binding EGF-like growth factor (HB-EGF), and basic fibroblast growth factor (bFGF) increased betacelullin mRNA levels. Only 2 of 15 normal samples and 1 of 10 cancer samples failed to exhibit the betacellulin transcript. Densitometric analysis of the autoradiographs revealed a 7.5-fold increase in betacellulin mRNA levels in the cancer tissues by comparison with the normal tissues. By in situ hybridization, the duct-like cancer cells exhibited many betacellulin mRNA in situ hybridization grains. These findings indicate that human pancreatic cancer cells express betacellulin in culture and in vivo, and suggest that this EGF-like ligand may participate in aberrant autocrine and paracrine activation of the EGF receptor in human pancreatic cancer.