At the onset of metamorphosis in Drosophila melanogaster, the steroid hormone 20-OH ecdysone induces a small number of early and early-late puffs in the polytene chromosomes of the third-instar larval salivary gland whose activity is required for regulating the activity of a larger set of late puffs. Most of the corresponding early and early-late genes have been found to encode transcription factors that regulate a much larger set of late genes. In contrast, we describe here the identification of an ecdysone-regulated gene in the 62E early-late puff, denoted D-spinophilin, that encodes a protein similar to the mammalian protein spinophilin/neurabin II. The D-spinophilin protein is predicted to contain a highly conserved PP1-binding domain and adjacent PDZ domain, as well as a coiled-coil domain and SAM domain, and belongs to a family of related proteins from diverse organisms. Transcription of D-spinophilin is correlated with 62E puff activity during the early stages of metamorphosis and is ecdysone-dependent, making this the first member of this gene family shown to be regulated by a steroid hormone. Examination of the dynamic patterns of D-spinophilin expression during the early stages of metamorphosis are consistent with a role in central nervous system metamorphosis as well as a more general role in other tissues. As D-spinophilin appears to be the only member of this gene family in Drosophila, its study provides an excellent opportunity to elucidate the role of an important adaptor protein in a genetic model organism.