Mos, the c-mos proto-oncogene product, is a key regulator of cell cycle progression. Recently, rapid turnover of Mos in an early stage of meiotic maturation of Xenopus oocytes was found to be mediated by the ubiquitin pathway, but the protease responsible for its breakdown was not identified. In the present study, we found that 35S-labeled Mos synthesized in an in vitro transcription/translation system was degraded ATP- and time-dependently by the 26S proteasome, but not by the 20S proteasome, in the presence of a ubiquitin-ligation system. The 26S proteasome did not degrade a mutant Mos in which Ser3 was replaced by Asp3 that is metabolically stable in oocytes, indicating a similarity in the proteolytic events in vivo to those observed in vitro in the present work. This is the first demonstration that the proteasome catalyzes the ATP-dependent degradation of a naturally occurring, short-lived oncoprotein by the ubiquitin pathway. This finding suggests that the proteasome may regulate the intracellular stability of various oncoproteins.