The neural cell adhesion molecule L1 has recently been shown to be expressed in pancreatic adenocarcinoma (PDAC) cells. In this report, we demonstrate that L1 is expressed by moderately- to poorly-differentiated PDAC cells in situ, and that L1 expression is a predictor of poor patient survival. In vitro, reduced reactivity of an anti-L1 carboxy-terminus-specific antibody was observed in the more poorly differentiated fast-growing (FG) variant of the COLO357 population, versus its well-differentiated slow-growing (SG) counterpart, even though they express equivalent total L1. The carboxy-terminus of L1 mediates binding to the MAP kinase-regulating protein RanBPM and mutation of T1247/S1248 within this region attenuates the expression of malignancy associated proteins and L1-induced tumorigenicity in mice. Therefore, we reasoned that the differential epitope exposure observed might be indicative of modifications responsible for regulating these events. However, epitope mapping demonstrated that the major determinant of binding was actually N1251; mutation of T1247 and S1248, alone or together, had little effect on C20 binding. Moreover, cluster assays using CD25 ectodomain/L1 cytoplasmic domain chimeras demonstrated the N1251-dependent, RanBPM-independent stimulation of erk phosphorylation in these cells. Reactivity of this antibody also reflects the differential exposure of extracellular epitopes in these COLO357 sublines, consistent with the previous demonstration of L1 ectodomain conformation modulation by intracellular modifications. These data further support a central role for L1 in PDAC, and define a specific role for carboxy-terminal residues including N1251 in the regulation of L1 activity in PDAC cells.