N-myc gene amplification is the most significant negative prognostic factor in pediatric neuroblastoma. Normal expression of the human N-myc oncogene is controlled at both transcriptional and post-transciptional levels. High levels of Myc protein appear to down-regulatemyc transcription, and evidence favors a common autoregulatory control mechanism for all myc gene family members. We have shown previously that a transfected N-myc promoter can be down-regulated by transfected c-myc in single-copy but not amplified lines, suggesting that autoregulation is intact only in single copy tumors. To test the hypothesis that loss of autoregulation is a prelude to amplification and to define cis-acting elements which mediate autoregulation, we transfected a series of N-myc 5′ promoter-deletion constructs into additional human neuroblastoma cell lines carrying one or multiple copies of N-myc. As noted previously, overexpression of c-Myc resulted in diminution of reporter activity in single-copy but not amplified lines. Using a series of promoter-deletion minigenes, we have localized an element necessary for autoregulation to within 192 bp upstream from the transcription start site. Surprisingly, however, the endogenous N-myc gene was down-regulated in the presence of forced c-Myc expression in both single-copy and amplified lines. Thus, two cis-acting sites are involved in autoregulation: one near the transcription start site, and another beyond the promoter region defined by our constructs. These studies represent the first systematic investigation of autoregulation in neuroblastoma, and indicate that single-copy lines produce insufficient N-Myc protein to activate downstream effectors of autosuppression; the autoregulatory loop is otherwise intact. Amplified cell lines, in contrast, appear to have lost a component of autoregulation.