Hormonal control of insect growth and metamorphosis by neuroendocrine system is well established, yet the cellular mechanism underlying the synthesis and release of insect hormones is not clearly understood. Here we report that a novel enhancer detection line, rutlethal-1096, failed to molt from second to third instar larvae, thus showing a lethal phenotype. Molecular cloning indicated that the enhancer detector was inserted in the proximal promoter region of the rut-encoded adenylyl cyclase (AC) gene. Similarly, the known rut alleles, rut1 and rut1084, were also found to be affected mildly in their larval and pupal development. The enriched expression of the reporter gene lacZ was detected in the ring gland that is an insect larval glandular organ secreting various hormones involved in ecdysis. Taken together, our data suggest the rut-encoded AC plays a vital role in larval molting, probably by regulating the function of ring glands.