AbstractTheLarval serum protein‐2gene (Lsp‐2) ofDrosophila melanogasterencodes one of the major hexameric haemolymph proteins of third‐instar larvae and a major component of adult serum. Regulated transcription ofLsp‐2results in high‐level, ecdysone‐stimulated expression throughout the larval fat body and low‐level, spatially restricted expression in the adult fat cells. To localize cis‐acting regulatory se quences responsible for the stage‐ and tissue‐specific activity ofLsp‐2, the expression ofLsp‐2–IacZfusion genes was studied byPelement‐mediated germline transformation ofDrosophila. A 230 base pair larval enhancer, which includes an ecdysone response element (EcRE), specifically targets gene activity to the larval fat body. Although the adult mode ofLsp‐2expression depends on the larval enhancer, additional negative regulatory elements dictate both tissue‐specificity and unique spatial restriction within the adult fat body. Implications of these findings for the identification of fat body‐specific gene regulatory uni