N-Alkylated polyamine analogues have been shown to exert antiproliferative effects in several tumor models, with the bis-ethyl derivatives exerting the greatest suppression of polyamines by virtue of down-regulation of the polyamine biosynthetic enzymes. Pancreatic adenocarcinoma presents a challenge both clinically and experimentally due to its inherent resistance to conventional therapy, which results in its having the worst 5-year survival rate of all cancers. We have previously shown that N1,N12-bis(ethyl)spermine (BESPM) is much more potent than the polyamine enzyme inhibitor alpha-difluoromethylornithine (DFMO) against pancreatic adenocarcinoma cell lines. In the present study, we compared the biochemical and antiproliferative effects of two N-alkylated polyamine analogues, N1,N14-bis(ethyl)homospermine (BEHSPM) and N1,N11-bis(ethyl)norspermine (BENSPM) in two human pancreatic ductal adenocarcinoma cell lines, PANC-1 (poorly differentiated) and BxPC-3 (moderately well-differentiated), and in the WD PaCa (well-differentiated ductal) hamster cell line. BENSPM displayed greater antiproliferative activity in the human pancreatic cancer cell lines, whereas BEHSPM was more potent in the hamster cell line. Both BEHSPM and BENSPM suppress the activity of the major biosynthetic enzymes ornithine decarboxylase and S-adenosylmethionine decarboxylase. However, the induction of polyamine depletion in the human cell lines was only modest for BENSPM and minimal for BEHSPM, which suggests that the substantial antiproliferative activity of these analogues may result from mechanisms other than polyamine depletion. The somewhat greater polyamine depletion seen following treatment with BENSPM is thought to result from its striking induction of spermidine/spermine N1-acetyltransferase. The biochemical and antiproliferative activity of BENSPM makes it an attractive agent for further preclinical and clinical development, especially in pancreatic cancer.