The developmentally programmed cell death of abdominal intersegmental muscles in the tobacco hawk-moth Manduca sexta is coincident with a 10-fold induction of the polyubiquitin gene as a hormonally regulated event (Schwartz, L. M., Myer, A., Kosz, L., Engelstein, M., and Maier, C. (1990) Neuron 5, 411-419). Solid phase immunochemical assays measuring intersegmental muscle pools of free and conjugated ubiquitin reveal that the induction of polyubiquitin mRNA is accompanied by a proportional increase in total ubiquitin polypeptide. Ubiquitin conjugate pools increase 10-fold at eclosion, during which loss of muscle protein mass is maximum. A smaller but measurable increase in ubiquitin conjugates is observed earlier in pupal development coincident with a modest enhanced degradation of myofibrillar proteins. Accumulation of ubiquitin conjugates is accompanied by induction in the pathway for polypeptide ligation, including the activating enzyme (E1), several carrier protein (E2) isoforms, and ubiquitin:protein isopeptide ligase (E3). Both accumulation of ubiquitin polypeptide and the enzymes of the conjugation pathway are subject to regulation by declining titers of the insect molting hormone 20-hydroxyecdysone, which signals onset of programmed cell death in the intersegmental muscles. Thus, programmed cell death within the intersegmental muscles is accomplished in part by stimulation of the ubiquitin-mediated degradative pathway through a coordinated induction of ubiquitin and the enzymes responsible for its conjugation to yield proteolytic intermediates. This suggests enzymes required for ubiquitin conjugation may represent additional genes recruited for developmentally programmed death.