The regulation of the Drosophila melanogaster hsp23 gene by heat shock and ecdysterone has been analysed by measuring activities of hsp--Escherichia coli beta-galactosidase hybrid genes in transfected hormone-sensitive D. melanogaster cells. Mutation analysis identified multiple, distinct promoter elements. A sequence element, which also occurs in the promoters of several other developmentally regulated Drosophila genes, is present in regions of the hsp23 promoter that are essential for its ecdysterone, but not its heat-regulated activity; this element may represent a binding site for an ecdysterone--receptor complex. Mutant promoters that can be activated only by heat shock or by hormone have been constructed. Thus the two types of regulation of the hsp23 gene can function independently of each other.