The notch gene family encodes transmembrane receptors that regulate cell differentiation by interacting with surface ligands on adjacent cells. Previously, we demonstrated that tumor necrosis factor-alpha (TNF) induces neuroendocrine (NE) cell differentiation in H82, but not H526, undifferentiated small cell lung carcinoma lines. We now test the hypothesis that TNF mediates NE cell differentiation in part by altering Notch gene expression. First, using RT-PCR in parallel with induction of gene expression for the NE-specific marker DOPA decarboxylase (DDC). Second, we treated H82 and H526 with notch-1 antisense versus sense oligodeoxynucleotides. Using quantitative RT-PCR and western analyses we demonstrate that DDC mRNA and protein are increased in H82 by notch-1 antisense, whereas notch-1 mRNA and activated Notch-1 protein are decreased. mRNA for HES-1, a transcription factor downstream from activated Notch, is also decreased by notch-1 antisense in H82 but not H526. After 7 days of notch1-antisense NCAM-immunoreactivity is induced in H82 but not H526. Third, we generated transgenic mice bearing notch-1 driven by the neural/NE-specific calcitonin promoter, which express activated Notch-1 in developing lung epithelium. Newborn NotchCal mouse lungs have high levels of HES-1 mRNA, reflecting increased activated Notch, compared with wild-type. NotchCal lungs have decreased: CGRP+ and PGP9.5+ NE cells; GRP, CGRP, and DDC mRNA levels compared to normal littermates. Cumulatively, these observations provide further support for a role for Notch-1 signaling in regulating pulmonary NE cell differentiation.