The Sgs-3 gene of Drosophila melanogaster exhibits a tightly regulated pattern of expression governed by two functionally equivalent elements within 1 kb of the gene, each of which is sufficient to confer third-instar salivary gland-specific transcription. In this report we describe a detailed functional analysis of one of these, the proximal element. To determine the nucleotides responsible for specific expression, we have introduced mutations into the proximal element and then assessed the effects of each alteration on expression in the developing animal. We have identified six particularly important base pairs which are located in two regions separated by nonessential sequences. These base pairs, along with some surrounding sequence, are conserved within the upstream regions of the three glue genes at 68C. Nearly identical groups of base pairs can be found upstream of the other glue genes which have been cloned. This analysis has allowed us to derive a consensus sequence, which we believe contains binding sites for two different factors which interact to direct third-instar salivary gland-specific expression.The Sgs-3 gene of Drosophila melanogaster encodes a glycoprotein component of the secretion produced in the salivary glands of the third-instar larva, which is expelled just prior to pupariation (1, 18). This "glue" serves to affix the pupa to a surface for the duration of metamorphosis (8). The Sgs-3 gene is one of three glue genes clustered at 68C on the left arm of the third chromosome (9, 32). Two other of the eight known glue genes, Sgs4 at 3C and Sgs-5 at 90BC, have also been cloned (34). The Sgs-3 gene exhibits a tightly regulated pattern of expression. It is abundantly transcribed only in a single cell type, of a single tissue, during a single 30-h period of development: the secretory cells of the salivary gland in the mid-to late third larval instar. Our interest has been in understanding how this extraordinarily specific expression pattern is achieved.Two trans-acting regulators of this gene have been previously described (see reference 33 for a review). The steroid hormone ecdysterone must be present at the time of the second molt, or no Sgs-3 mRNA is produced (14). In addition, particular mutations at the 2B5 locus, such as that of the nonpupariating mutant npr-13, fail to produce Sgs-3 mRNA (5). Whether either of these factors acts directly at the Sgs-3 locus is not yet known.The required cis-acting sequences have been investigated in our laboratory and by others (for reviews, see references 29 and 33). An element located within 130 bp of the transcription start site (the proximal element) is sufficient for correctly regulated but low-level expression of this gene (54). A second element, located between -130 and -629 bp relative to the transcription start site (the distal element), is also sufficient for regulated low-level expression (43). When these elements are both present, a 20-fold enhancement of the level of expression is seen. Within the proximal element,