Expression of the human N-myc oncogene is normally restricted to developing epithelial tissues and pre-B cells at a specific stage of differentiation. We have localized cis-acting sequences which appear responsible for silencing expression in cell types which do not contain detectable N-myc mRNA by Northern or RT-PCR analysis. We have determined that 5′ promoter sequences within 2 kb of the transcription initiation site allow reporter expression in human cell lines derived from a wide variety of tissues, regardless of endogenous N-myc mRNA expression. In contrast, inclusion of the contiguous 902 bp, containing the remainder of the untranslated first exon and extending into the first intron, restores a physiologic expression pattern, in that reporter constructs are active only in cells which express endogenous N-myc. Using transient transfection of reporters bearing progressive 3′ deletions of this larger construct, we localized an element responsible for complete silencing of N-myc expression in non-expressing cells to a 116 bp region within intron 1 (nt +598 to +714). This 116 bp, element alone, however, is not sufficient to silence transcription from a heterologous promoter, indicating that additional sequences play a role. A second site within the first exon (nt+151 to +174) also down-regulates expression, as reporter activity in non-expressing cells is diminished by 30-60% relative to that of the 5′ promoter alone. We have performed nuclear run-on analyses to determine the mechanism(s) of this tissue-specific expression. All N-myc non-expressing cell lines examined show active transcription across the gene, indicating that regulation of expression occurs by a post-transcriptional mechanism. This mechanism of post-transcriptional control adds another level of complexity to the regulation of the human N-myc oncogene. Identification and characterization of the trans-acting factor(s) involved in this process may reveal components which could be exploited in the development of improved therapeutic strategies for several malignancies which currently carry a poor prognosis.