A derivative of ubiquitin in which amino groups were blocked by reductive methylation was used to study the possible role of the ubiquitin pathway in the cell cycle-programmed degradation of cyclin. It was shown previously that methylated ubiquitin can be efficiently ligated to protein substrates, but cannot form polyubiquitin chains. In the well-characterized ubiquitin-dependent proteolytic system from reticulocytes, it was found that rates of protein breakdown obtained with methylated ubiquitin are generally slower than those with ubiquitin; and thus, this derivative can be used, in some cases, as an inhibitor of ubiquitin-dependent protein degradation. The addition of methylated ubiquitin to a cell-free system from fertilized clam oocytes inhibited the degradation of both cyclins A and B. That this was due to specific interference with ubiquitin function was indicated by the observation that the supplementation of excess ubiquitin completely overcame the inhibitory action of methylated ubiquitin on cyclin degradation. These findings suggest that polyubiquitin chain formation is required for cyclin degradation.