The sensitivity of three human colon adenocarcinoma cell lines (LoVo, LS174T, and SW1116) and a human pancreatic adenocarcinoma cell line (Hs766T) to a recombinant ricin A chain-antitransferrin receptor immunotoxin was studied. In addition, the carboxylic ionophore monensin was used in conjunction with the immunotoxin to determine the possibility of increased cytotoxicity without loss of specificity. The immunotoxin, 454A12-rRTA, is composed of the monoclonal antibody 454A12 directed against transferrin receptor and of ricin A chain, which was produced by recombinant DNA techniques. In 18 h dose-response cytotoxicity assays, the median inhibitory dose (ID50) against LoVo, LS174T, and SW1116 was found to be 3 ± 10–10, 3.6 ± 10, and 3.6 ± 10–10M, respectively; in the same assay, the ID50for Hs766T was found to be 4 ± 10–10M. In the presence of monensin, the ID50for the adenocarcinoma cell lines was reduced 9-fold, 28-fold, and 5-fold, respectively. In cytotoxic kinetic assays, 50% of control protein inhibition was reached in immunotoxin-treated LS174T cells 12-fold faster in the presence of monensin than in its absence. Immunotoxin-treated LoVo cells reached 50% inhibition of control protein synthesis fivefold faster in the presence of monensin than in its absence. Furthermore, no toxicity of immunotoxin or potentiation by monensin was observed in either a control cell line (Swiss albino mouse 3T6) treated with specific immunotoxin or with a control immunotoxin assay. These results show the in vitro specificity and selectivity of 454A12-rRTA immunotoxin for human gastrointestinal and pancreatic cancer cell lines.