Reports on hormone receptor expression of pancreatic cancer (PaCa) cells and treatment responses to antihormonal therapy are conflicting. We examined estrogen receptor (ER) expression in PaCa cells and investigated its function in estrogen-mediated cell proliferation.Protein levels of ERα and ERβ in 8 human PaCa lines were detected by Western blot analysis. Cell proliferation was measured by sulforhodamine B analysis. ER modulators included diethylstilbestrol (DES), estradiol (E2), 4-hydroxytamoxifen (Tam), genistein (Gen), and Coumestrol (Coum).ERα levels were detected in all eight, and ERβ in seven cell lines. ERβ/ERα ratio ranged from 0.4 to 111 (median: 6.4, >5 in seven lines). Median maximal growth stimulation (in %, observed at 20 to 200 nM) was 19 (DES), 39 (E2), 20 (Tam), 22 (Gen), and −9 (Coum); median maximal inhibition (at 40 to 60 μM) was 59 (DES), 36 (E2), 25 (Tam), 43 (Gen), and 50 (Coum). The extent of E2 and Gen stimulatory effects correlated with the ERβ/ERα ratio (Kendall’s τ: 0.714, P = 0.024), but not ERα or ERβ levels alone. Only Coum-induced inhibition correlated with the ERβ/ERα ratio (P = 0.006) and with ERα expression (r = 0.753, P = 0.03). Gemcitabine-induced PaCa cytotoxicity (at IC 40 ) was significantly reduced by E2, Gen, and Coum.PaCa proliferation in vitro is highly estrogen sensitive, and in contrast to other reports, ERs are frequently expressed. In 7/8 cell lines, ERβ expression outweighs ERα expression. The impact of the ERβ/ERα ratio on estrogen-mediated growth stimulation and reduced cytotoxicity at physiological concentrations may have clinical implications on PaCa therapy.