Overexpression of autocrine growth factors and their receptors has been reported in many human cancers. The study of autocrine-regulated pathways using in vitro culture systems can be hindered by the presence of fetal bovine serum in culture medium. A human pancreatic cancer cell line (HPAF) was slowly weaned from its dependence on fetal bovine serum and subsequently maintained in serum-free conditions. Growth factor secretion studies showed that production of autocrine growth factors such as transforming growth factor alpha, gastrin-releasing peptide, and insulin-like growth factor I from weaned cells increased three times compared with nonweaned cells (p < 0.01). The epidermal growth factor and gastrin-releasing peptide receptor densities were also increased in weaned cells (2 times and 2.5 times, respectively, p < 0.05). The proliferation of weaned cells cultured continuously in the same medium was significantly greater than of nonweaned cells (p < 0.05). Collectively, these data indicate that weaned pancreatic cancer cells can proliferate in the absence of serum by up-regulating autocrine pathways.