Neuroblastoma is a childhood tumor that originates from the sympathetic nervous system. The tumor cells have embryonic features, presumably as a consequence of an impaired capacity to respond to signals and transcriptional control mechanisms operating during normal differentiation. Two basic helix‐loop‐helix transcription factors, human achaete‐scute homologue‐1 (HASH‐1) and hairy/enhancer of split homologue‐1 (HES‐1), are crucial for proper development of some neuronal cells. Here, their potential roles during sympathetic differentiation of human neuroblastoma cells have been investigated. In all tested protocols for induction of differentiation of SH‐SY5Y and SK‐N‐BE(2) neuroblastoma cells, HASH‐1 expression was rapidly decreased with a concomitant, often transient, increase in HES‐1 expression. In gel mobility shift assays, using extracts from neuroblastoma cells, HES‐1 bound to an oligonucleotide corresponding to a sequence in the HASH‐1 promoter including the so‐called N‐box, suggesting that the transiently increased HES‐1 activity in differentiating neuroblastoma cells is involved in down‐regulation of HASH‐1. Constitutive expression of the intracellular domain of Notch1, which activates the HES‐1 promoter in SH‐SY5Y cells, inhibited spontaneous and induced morphological differentiation of these neuroblastoma cells. Our data show that functional sympathetic neuronal differentiation of neuroblastoma cells is associated with transient activation of HES‐1 and down‐regulation of HASH‐1 expression. Int. J. Cancer 88:401–410, 2000. © 2000 Wiley‐Liss, Inc.