In the moth Manduca sexta, intersegmental muscles (ISMs) undergo rapid programmed cell death (PCD) within 48 hr of adult emergence. ISM PCD involves ubiquitin-dependent proteasomal degradation accompanied by the down-regulation of expression of actin genes and the up-regulation of degradative gene expression such as ubiquitin. Hemin chloride and N-acetyl-leu-leu-norleucinal (ALLN), both inhibitors of proteasomal activity, administered before adult emergence delayed PCD for up to 5 days in ISMs maintained from the larval stage, such as the dorsal internal medial muscle in abdominal segment 4 (DIM-A4). ISMs that developed during metamorphosis from respecified larval muscles such as the DIM-A2 were less dramatically affected. The increase in polyubiquitinated proteins and the decrease in actin mRNA expression accompanying maintained ISM PCD were delayed after inhibitor application. No changes were detected in respecified ISMs. These results reveal a regulatory role for proteasomal activity in an early stage of maintained ISM cell death. Developmental Dynamics 233:445–455, 2005. © 2005 Wiley-Liss, Inc.