The aims of this study were to determine the effects of (a) combining the epidermal growth factor receptor (EGFR) blocker (erlotinib) and the cyclooxygenase-2 inhibitor (celecoxib) on cell growth and apoptosis in human pancreatic cancer cell lines, (b) baseline EGFR expression on the potentiation of erlotinib-induced apoptosis by celecoxib, and (c) the effects of the combination on the expression of the COX-2, EGFR, HER-2/neu, and nuclear factor-kappaB (NF-kappaB). Baseline expression of EGFR was determined by Western blot analysis in five human pancreatic cancer cell lines. BxPC-3, PANC-1, and HPAC had high EGFR and MIAPaCa had low EGFR. Cells were grown in culture and treated with erlotinib (1 and 10 micromol/L), celecoxib (1 and 10 micromol/L), and the combination. Growth inhibition was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and apoptosis was assayed by ELISA. Reverse transcriptase-PCR was used to evaluate COX-2 and EGFR mRNA. EGFR, COX-2, and HER-2/neu expression was determined by Western immunoblotting. Electrophoretic mobility shift assay was used to evaluate NF-kappaB activation. Growth inhibition and apoptosis were significantly (P < 0.05) higher in BxPC-3, HPAC, and PANC-1 cells treated with celecoxib and erlotinib than cells treated with either celecoxib or erlotinib. However, no potentiation in growth inhibition or apoptosis was observed in the MIAPaCa cell line with low expression of the EGFR. Significant down-regulation of COX-2 and EGFR expression was observed in the BxPC-3 and HPAC cells treated with the combination of erlotinib (1 micromol/L) and celecoxib (10 micromol/L) compared with celecoxib- or erlotinib-treated cells. Celecoxib significantly down-regulated HER-2/neu expression in BxPC-3 and HPAC cell lines. Significant inhibition of NF-kappaB activation was observed in BxPC-3 and HPAC cell lines treated with erlotinib and celecoxib. (a) Celecoxib can potentiate erlotinib-induced growth inhibition and apoptosis in pancreatic cell lines, (b) high baseline EGFR expression is a predictor of this potentiation, and (c) the down-regulation of EGFR, COX-2, and HER-2/neu expression and NF-kappaB inactivation contributes to the potentiation of erlotinib by celecoxib.