When human esophageal cancer cells were transfected with the human interferon-beta (hIFN-beta) gene entrapped in cationic multilamellar liposomes, the growth of all cancer cells tested was suppressed in a dose-dependent manner. The 50% inhibitory concentration (IC50) of the hIFN-beta gene entrapped in the liposomes ranged from 16 to 176 ng plasmid DNA/ml culture medium. Among the 10 cell lines examined, NUEC3, NUEC4, TE-3 and WSSC cell lines were highly susceptible to transfection with this gene entrapped in the liposomes. The IC50 values of the hIFN-beta gene entrapped in the liposomes with respect to cell growth were positively-correlated with those of exogenous cytokine hIFN-beta, suggesting that the antiproliferative effect of hIFN-beta gene entrapped in the liposomes can be mainly ascribed to the function of hIFN-beta produced by cells transfected with the gene. Two days after transfection with the liposome-entrapped gene, the concentration of hIFN-beta secreted into the medium was determined. Even though the level of hIFN-beta observed in the medium was lower than that of the IC50 of exogenously added hIFN-beta, the inhibitory potency of the hIFN-beta gene entrapped in the liposomes on the cell growth was remarkable. When the esophageal cancer cells were treated with 5-fluorouracil (5-FU) in the presence of a low concentration of liposome-entrapped-gene, the rate of growth inhibition of these cells increased over that caused by either 5-FU or hIFN-beta gene entrapped in the liposomes alone. All these data suggest that combination therapy with the hIFN-beta gene entrapped in cationic multilamellar liposomes and the anticancer drug 5-FU would be beneficial for preoperative treatment of carcinoma of the esophagus.