We have shown that covalent conjugation of ubiquitin to proteins is temperature-sensitive in the mouse cell cycle mutant ts85 due to a specifically thermolabile ubiquitin-activating enzyme (accompanying paper). We show here that degradation of shortlived proteins is also temperature sensitive in ts85, in contrast to wild-type and revertant cells. While more than 70% of the prelabeled abnormal proteins (containing amino acid analogs) or puromycyl peptides are degraded within 4 hr at the permissive temperature in both ts85 and wild-type cells, less than 15% are degraded in ts85 cells at the nonpermissive temperature. Degradation of abnormal proteins and puromycyl peptides in both ts85 cells and wild-type cells is nonlysosomal and ATP-dependent. lmmunochemical analysis shows a strong and specific reduction in the levels of in vivo labeled ubiquitin-protein conjugates at the nonpermissive temperature in ts85 cells. Degradation of normal, shortlived proteins is also specifically temperature sensitive in ts85. We suggest that the contribution of ubiquitin-independent pathways to the degradation of short-lived proteins in this higher eucaryotic cell is no more than lo%, and possibly less.