Normal expression of the N-myc gene is restricted to fetal epithelial tissues and adult pre-B cells. The gene is amplified and overexpressed in a subset of neuroblastomas. Elucidation of the normal mechanisms of N-myc gene regulation may lead to more directed therapies for this tumor. Using N-myc promoter-reporter transfections, we and others noted that the 5′ portion of the promoter is active in many cell types, including those lacking endogenous N-myc expression. In addition, nuclear run-on analyses using single-stranded targets demonstrate transcription across the entire N-myc gene even in cell types which do not express endogenous N-myc, suggesting that post-transcriptional mechanisms regulate expression. We localized a 116 bp element within intron 1 which is sufficient to direct expression to only those cell types in which the endogenous N-myc gene is active. This intron element alone can decrease expression from a heterologous promoter (pSV40) in N-myc non-expressing cell types to 1-10% of that in N-myc expressing cells. This element functions to decrease expression only when placed within the transcriptional unit, but not when located upstream from the transcription start site. Surprisingly, this intron element decreases reporter activity in N-myc non-expressing cells equally well regardless of its orientation. This intron element destabilizes luciferase transcripts derived from transfected reporter plasmids in N-myc non-expressing U937 cells (T1/2 = 2.9 ± 0.5 hrs. for pN-myc intron-luciferase vs. 16.7 ± 3 hrs. for pN-myc luciferase). Taken together, these data indicate that a major control mechanism restricting human N-myc expression to specific tissues is posttranscriptional, and that an element within the first intron acts to decrease expression by destabilizing unprocessed RNA.