Neuroblastoma cell lines typically exhibit multiple cell phenotypes, counterparts of those comprising the embryonic neural crest. Expression of the MYCN gene, usually amplified in cell lines, differs markedly among the various differentiation phenotypes. Whereas neuroblastic (N-type) and stem cell (I-type) sublines have abundant MYCN RNA and protein, S-type cells (nonneuronal neural crest precursors) have a 4- to 9-fold lower level of cytoplasmic mRNA and a 2- to 36-fold lower protein content. N and S sublines with chromosomally integrated MYCN genes have similar gene copy numbers. Thus, in these S cells, MYCN expression is downregulated. Nuclear run-on and mRNA stability assays have revealed similar transcription rates and mRNA half-lives in N and S cells from two cell lines, indicating that downregulation occurs posttranscriptionally prior to mRNA degradation in the cytoplasm. S-type cells derived from double minute chromosome-containing lines show 4- to 10-fold lower gene copy numbers than N counterparts. Experimental induction of differentiation to neuronal/neuroendocrine or to S-type cells results in a marked reduction of MYCNexpression and, in double minute chromosome-containing N-type sublines, in gene loss as well. Malignant potential as indicated by soft agar growth capacity and tumor formation in nude mice is markedly diminished in S cells and, generally, is directly proportional to MYCN mRNA levels. The most plausible relationship suggested by our data is that MYCN expression, regulated by cell lineage and/or differentiation state, directly modulates the malignant potential of human neuroblastoma cells.