Nuclear oncoproteins are among the most rapidly degraded intracellular proteins. Previous work has implicated the ubiquitin-mediated proteolytic system in the turnover of short-lived intracellular proteins as a class. In the present study, we have evaluated the potential role of the ubiquitin system in the degradation of the specific nuclear oncoproteins encoded by the MYCN gene. The oncoproteins were synthesized in vitro by transcription of an MYCN cDNA, and translation of the resulting single species of mRNA in the presence of 35S-methionine. Degradation of labeled proteins was monitored in a cell-free system derived from rabbit reticulocytes. ATP stimulated the degradation of the MYCN protein more than 10 fold. ATP-dependent degradation was completely inhibited by neutralizing antibody directed against E1, the first enzyme in the ubiquitin-mediated proteolytic cascade. Moreover, ATP-dependent degradation in E1-depleted lysate could be restored by the addition of affinity-purified E1. These data suggest that the ubiquitin system mediates the ATP-dependent degradation of MYCN oncoproteins in vitro, and that these proteins possess signals that target them for rapid turnover by this proteolytic pathway. Although MYCN in neuroblastomas is activated primarily by amplification, it may be activated by other mechanisms in aggressive tumors with a single copy of MYCN. Mutations in protein coding sequence that alter the signals for recognition by protein degradation systems represent a potential mechanism by which oncogene activation might occur.