A major prognostic marker for neuroblastoma (Nb) is N-myc gene amplification, which predicts a poor clinical outcome. We sought genes differentially expressed on a consistent basis between multiple human Nb cell lines bearing normal versus amplified N-myc, in hopes of finding target genes that might clarify how N-myc overexpression translates into poor clinical prognosis. Using differential display, we find the previously described growth-inhibitory gene Ndrg1 is strongly repressed in all tested Nb cell lines bearing N-myc amplification, as well as in a neuroepithelioma line with amplified c-myc. Overexpression of N-myc in non-amplified Nb cells leads to repression of Ndrg1, as does activation of an inducible c-myc transgene in fibroblasts. Conversely, N-myc downregulation in N-myc-amplified Nb cells results in re-expression of the Ndrg1, and stimuli known to induce Ndrg1 do so in Nb cells while simultaneously down-regulating N-myc. Relevant to these results, we demonstrate an in vitro interaction of Myc protein with the Ndrg1 core promoter. We also find that Ndrg1 levels increase dramatically during in vitro differentiation of two cell lines modeling neural and glial development, while c- and N-myc levels decline. Our results combined with previous information on the Ndrg1 gene product suggest that downregulation of this gene is an important component of N-Myc effects in neuroblastomas with poor clinical outcome. In support of this notion, we find that re-expression of Ndrg1 in high-Myc Nb cells results in smaller cells with reduced colony size in soft-agar assays, further underscoring the functional significance of this gene in human neuroblastoma cells.