Among the various forms of human lung cancer, small cell lung cancer (SCLC) exhibits a characteristic neuroendocrine (NE) phenotype. Neural and NE differentiation in SCLC depend, in part, on the action of the basic-helix-loop-helix (bHLH) transcription factor human achaete-scute homologue-1 (hASH1). In nervous system development, the Notch signaling pathway is a critical negative regulator of bHLH factors, including hASH1, controlling cell fate commitment and differentiation. To characterize Notch pathway function in SCLC, we explored the consequences of constitutively active Notch signaling in cultured SCLC cells. Recombinant adenoviruses were used to overexpress active forms of Notch1, Notch2, or the Notch effector protein human hairy enhancer of split-1 (HES1) in DMS53 and NCI-H209 SCLC cells. Notch proteins, but not HES1 or control adenoviruses, caused a profound growth arrest, associated with a G₁ cell cycle block. We found up-regulation of p21^waf1/cip1 and p27^kip1 in concert with the cell cycle changes. Active Notch proteins also led to dramatic reduction in hASH1 expression, as well as marked activation of phosphorylated extracellular signal-regulated kinase (ERK)1 and ERK2, findings that have been shown to be associated with cell cycle arrest in SCLC cells. These data suggest that the previously described function of Notch proteins as proto-oncogenes is highly context-dependent. Notch activation, in the setting of a highly proliferative hASH1-dependent NE neoplasm, can be associated with growth arrest and apparent reduction in neoplastic potential.