The opioid growth factor (OGF), [Met5]-enkephalin, and OGF receptor (OGFr) form an inhibitory axis regulating the growth of human pancreatic cancer. This study examined whether overexpression of OGFr decreases the growth of pancreatic cells in vitro. MIA PaCa-2 cells were transfected with OGFr cDNA, and six clonal lines were examined for protein expression and function. OGFr binding assays revealed a 2.3- to 5.6-fold increase in binding capacity from wild-type (WT) and empty vector (EV) controls; binding affinity was comparable in all groups. OGFr protein expression, as measured by immunohistochemistry and Western blotting, was enhanced in clonal cell lines compared to controls. Doubling times of OGFr clonal lines were 47-91% longer than in the WT/EV groups for all but one clonal line. DNA synthesis of cells overexpressing OGFr was diminished from the WT/EV groups by 28-52%. Addition of exogenous OGF further reduced (14-31%) the cell growth of clonal lines, and the effects of exogenous OGF were receptor-mediated. Exposure of cells overexpressing OGFr to naltrexone increased the cell number by up to 9.4-fold. OGF was identified as the only opioid peptide to depress cell replication in the transfected cell lines. Neutralization of endogenous OGF with antibodies to this peptide elevated the cell number in clonal cell lines. These data identify OGFr at the molecular level as integral to regulating the cell replication of human pancreatic cancer, and support treatment modalities that amplify OGFr in order to decrease the growth of these neoplasias.