During metamorphosis of the hawkmoth, Manduca sexta, ecdysteroids regulate the dendritic remodeling and programmed death of identified motoneurons. These changes contribute to the dramatic reorganization of behavior that accompanies metamorphosis. As a step toward elucidating cellular and molecular mechanisms by which ecdysteroids affect neuronal phenotype, we have investigated the responses of Manduca motoneurons to ecdysteroids in vitro. Following dendritic regression at the end of larval life, thoracic leg motoneurons placed in culture respond to ecdysteroids by an increase in branching complexity, similar to events in vivo. Growth cone structure is affected markedly by ecdysteroids. At pupation, a rise in ecdysteroids triggers the segment-specific death of proleg motoneurons: the same segmental pattern of death is observed when motoneurons from different segments are removed from the nervous system and exposed to ecdysteroids in vitro. These studies provide strong evidence that Manduca motoneurons are direct targets of steroid action and set the stage for further studies of the specific mechanisms involved.